Recombinant viral switches for the control of gene expression in plants

ABSTRACT

The invention describes a method of controlling a biochemical process or a biochemical cascade in plants utilizing a process of interaction between a heterologous DNA sequence in a transgenic plant, on one side, and a heterologous DNA sequence in a plant viral transfection vector, on the other. Optionally, the process of interaction further involves a low molecular weight component. The process of interaction makes the infection with a viral transfection vector a gene-“switch” which switches on a biochemical process or cascade of interest via various reactions such as nucleic acid recombination, replication, transcription, restriction, translation, protein folding, assembly, targeting, posttranslational processing, or enzymatic reaction. Further a process for producing a product in a transgenic plant and kit of parts for such a process is provided.

FIELD OF THE INVENTION

The present invention relates to a process of controlling a biochemicalprocess or biochemical cascade of interest in a plant according to thepreamble of claim 1. Moreover, the present invention relates to aprocess for producing a product in a transgenic plant by using theprocess of controlling a biochemical process or biochemical cascade ofinterest according to the invention. Further, the present inventionrelates to a kit-of-parts for performing the processes of the invention.The process of the invention allows for the selective control oftransgene expression in a transgenic plant whereby a biochemical processor biochemical cascade of interest previously non-operable in the plantmay be selectively switched on at any predetermined time.

BACKGROUND OF THE INVENTION

Controllable Transgene Expression Systems in Plants

One of the major problems in plant biotechnology is the achievement ofreliable control over transgene expression. Tight control over geneexpression in plants is essential if a downstream product of transgeneexpression is growth inhibitory or toxic, like for example,biodegradable plastics (Nawrath, Poirier & Somerville, 1994, Proc. Natl.Acad. Sci, 91,12760-12764; John & Keller, 1996, Proc. Natl. Acad. Sci.,93, 12768-12773; U.S. Pat. No. 6,103,956; U.S. Pat. No. 5,650,555) orprotein toxins (U.S. Pat. No. 6,140,075).

Existing technologies for controlling gene expression in plants areusually based on tissue-specific and inducible promoters and practicallyall of them suffer from a basal expression activity even when uninduced,i.e. they are “leaky”. Tissue-specific promoters (U.S. Pat. No.5,955,361;.WO09828431) present a powerful tool but their use isrestricted to very specific areas of applications, e.g. for producingsterile plants (WO9839462) or expressing genes of interest in seeds(WO00068388; U.S. Pat. No. 5,608,152). Inducible promoters can bedivided into two categories according to their inductionconditions—those induced by abiotic factors (temperature, light,chemical substances) and those that can be induced by biotic factors,for example, pathogen or pest attack. Examples of the first category areheat-inducible (U.S. Pat. No. 5,187,287) and cold-inducible (U.S. Pat.No. 5,847,102) promoters, a copper-inducible system (Mett et al., 1993,Proc. Natl. Acad. Sci., 90, 4567-4571), steroid-inducible systems(Aoyama & Chua, 1997, Plant J., 11, 605-612; McNellis et al., 1998,Plant J., 14, 247-257; U.S. Pat. No. 6,063,985), an ethanol-induciblesystem (Caddick et al., 1997, Nature Biotech., 16, 177-180; WO09321334),and a tetracycline-inducible system (Weinmann et al., 1994, Plant J., 5,559-569). One of the latest developments in the area of chemicallyinducible systems for plants is a chimaeric promoter that can beswitched on by glucocorticoid dexamethasone and switched off bytetracycline (Bohner et al., 1999, Plant J., 19, 87-95). For a review onchemically inducible systems see: Zuo & Chua, (2000, Current Opin.Biotechnol., 11, 146-151). Other examples of inducible promoters arepromoters which control the expression of patogenesis-related (PR) genesin plants. These promoters can be induced by treatment of the plant withsalicylic acid, an important component of plant signaling pathways inresponse to pathogen attack, or other chemical compounds(benzo-1,2,3-thiadiazole or isonicotinic acid) which are capable oftriggering PR gene expression (U.S. Pat. No. 5,942,662).

There are reports of controllable transgene expression systems usingviral RNA/RNA polymerase provided by viral infection (for example, seeU.S. Pat. No. 6,093,554; U.S. Pat. No. 5,919,705). In these systems, arecombinant plant DNA sequence includes the nucleotide sequences fromthe viral genome recognized by viral RNA/RNA polymerase. Theeffectiveness of these systems is limited because of the low ability ofviral polymerases to provide functions in trans, and their inability tocontrol processes other than RNA amplification.

The systems described above are of significant interest as opportunitiesof obtaining desired patterns of transgene expression, but they do notallow tight control over the expression patterns, as the inducing agents(copper) or their analogs (brassinosteroids in case ofsteroid-controllable system) can be present in plant tissues at levelssufficient to cause residual expression. Additionally, the use ofantibiotics and steroids as chemical inducers is not desirable for thelarge-scale applications. When using promoters of PR genes or viralRNA/RNA polymerases as control means for transgenes the requirements oftight control over transgene expression are also not fulfilled, ascasual pathogen infection or stress can cause expression. The tissue ororgan-specific promoters are restricted to very narrow areas ofapplications, since they confine expression to a specific organ or stageof plant development, but do not allow the transgene to be switched onat will.

Plant Viral Vectors and Their Use in the Field of Applied Plant Virology

Presently, there are three distinct major fields in the area of appliedplant virology: a) use of viruses as vectors for transgeneoverexpression; b) use of viruses as vectors for plant functionalgenomics, and c) use of viral components in the field of phytopathologyfor generating virus-resistant transgenic plants.

Plant viruses can serve as efficient tools for high level expression oftransgenes in host plant species. The use of transgenic plant virus infield does not seem to compromise any biosafety issues. For example,Animal and Plant Health Inspection Service, USDA, did not find anysignificant impact after field trials with genetically modified TMV(tobacco mosaic virus) and tobacco etch viruses containing heterologousgenes of pharmaceutical interest. As a result, two permissions wereissued in 1996 and 1998. Work has been conducted in the area ofdeveloping viral vectors for transferring foreign genetic material intoplant hosts for the purposes of expression (U.S. Pat. No. 4,885,248;U.S. Pat. No. 5,173,410). There are several patents which describe thefirst viral vectors suitable for systemic expression of transgenicmaterial in plants (U.S. Pat. No. 5,316,931; U.S. Pat. No. 5,589,367;U.S. Pat. No. 5,866,785). In general, these vectors can express foreigngenes from an additional subgenomic promoter (U.S. Pat. No. 5,466,788;U.S. Pat. No. 5,670,353; U.S. Pat. No. 5,866,785), as translationalfusions with viral proteins (U.S. Pat. No. 5,491,076; U.S. Pat. No.5,977,438) or from polycistronic viral RNA using IRES elements forindependent protein translation, also used herein, according to ANNEX Acorresponding to German Patent Application No 10049587.7. Carrington etal., (U.S. Pat. No. 5,491,076) describe the use of an endogenous viralprotease to cleave heterologous proteins from viral polyproteins.Another area of application for viral vectors is plant functionalgenomics. Della-Cioppa et al., (WO993651) describe the use of TMV-basedviral vectors for expression of plant cDNA libraries with the purpose ofsilencing endogenous genes.

Angell & Baulcombe (1997, EMBO J, 16, 3675-3684; WO9836083) describe aPVX-based system called “Amplicon™” designed for down-regulating thetargeted genes in plants. The same system in combination with Hc-Prothat suppresses transgene silencing in plants (Pruss et al., 1997, PlantCell, 9, 859-868; U.S. Pat. No. 5,939,541) is used for overexpression oftransgenes. U.S. Pat. No. 5,939,541 describes an approach based on usingthe 5′proximal region (booster sequence, including the Hc-Pro gene) ofthe potyvirus to enhance expression of any gene in plants. This sequencecan be stably integrated into the plant genome or delivered by a virus.It is worth mentioning that Hc-Pro has a pronounced pleiotropic effectand enhances the expression of both transgenes and endogenous plantgenes. Thus, these systems provide at best a quantitative improvement oftotal protein expression over existing processes. They do so byinfluencing many components of the protein production machinery by anunknown mechanism and in a hardly controlled manner.

There is an abundant literature including patent applications whichdescribe the design of virus resistant plants by the expression of viralgenes or mutated forms of viral RNA (e.g. U.S. Pat. No. 5,792,926; U.S.Pat. No. 6,040,496). It is also worth mentioning that an environmentalrisk is associated with the use of such plants due to the possibility offorming novel viruses by recombination between the challenging virus andtransgenic viral RNA or DNA (Adair & Kearney, 2000, Arch. Virol, 145,1867-1883).

Therefore, it is an object of the present invention to provide anenvironmentally safe process of controlling a biochemical process or abiochemical cascade of interest in a plant whereby the process orcascade may be selectively switched on at any predetermined time.

It is another object of this invention to provide a process forproducing a product in a transgenic plant wherein the production of theproduct may be selectively switched on after the plant has grown to adesired stage, whereby the process is environmentally safe and does notlead to the release of potentially hazardous functional transgenes inthe environment.

Another object of this invention is to provide a kit of parts forperforming such processes.

GENERAL DESCRIPTION OF THE INVENTION

These objects are achieved by a process according to claim 1. Morespecifically, these objects are achieved by a process of controlling abiochemical process or biochemical cascade of interest in a plant, saidprocess being characterized by comprising the following steps:

-   -   (a) introducing into the nuclear genome of the plant one or more        first heterologous DNA sequences,    -   (b) infecting the plant with at least one viral transfection        vector containing in its genome one or more second heterologous        DNA sequences, thus triggering a process of interaction in the        plant between    -   (i) one or more first heterologous DNA sequences of the nuclear        genome and/or expression products of the first heterologous DNA        sequences, and    -   (ii) one or more second heterologous DNA sequences of the        transfection vector and/or expression products of the second        heterologous DNA sequences, and    -   (iii) optionally one or more externally added low molecular        weight components,        thus switching on the biochemical process or cascade of interest        that was not operable prior to said interaction. Preferably,        said first heterologous DNA sequence(s) in the above processes        are of non-plant viral origin, i.e. do not originate from a        plant virus.

The present invention further provides a process of producing a productin a transgenic plant comprising the process of controlling abiochemical process or a biochemical cascade of interest in a plantaccording to the invention. In particular, the process further comprisesthe following steps:

-   -   (a) growing the transgenic plant to a desired stage, followed by    -   (b) infecting the plant with one or more vectors, and optionally        contacting the plant with one or more low molecular weight        components, thus switching on the biochemical process or cascade        necessary for the production of the product, said process or        cascade not being operable prior to said interaction, and    -   (c) producing the product in the plant,        whereby said vector is preferably a viral transfection vector.

Further a kit of parts is provided for the above processes comprising atransgenic plant or seeds thereof and a virus-based vector. Also, a kitof parts is provided comprising a transgenic plant and one or morevectors, whereby said vector(s) may give rise to one or more viraltransfection vectors in a plant. Said transgenic plant preferablycontains a first heterologous DNA sequence according to step (a) of theprocess of the invention.

Further, a vector for performing step (b) of the process of theinvention and a plant obtained or obtainable by the process of theinvention is provided.

According to the invention it is possible to selectively switch on abiochemical process or biochemical cascade in a transgenic plant byinfecting the transgenic plant with one or more viral transfectionvectors. The biochemical process or cascade is not operable in thetransgenic plant prior to the infection with the viral vector for lackof essential elements or functions necessary to perform the biochemicalprocess or cascade. Essential elements may be e.g. a promoter, an RNApolymerase, a transcription factor or the like. Essential functions maybe transcription, translation or enzymatic activity which is notoperable e.g. for lack of functional coupling of a promoter with adownstream sequence to be expressed. The biochemical process or cascadebecomes operable by a process of interaction triggered by the infection.The process of interaction in the plant requires one or more firstheterologous DNA sequences of the nuclear genome and/or expressionproducts of the first heterologous DNA sequences, and one or more secondheterologous DNA sequences of the transfection vector and/or expressionproducts of the second heterologous DNA sequences, and optionally one ormore externally added low molecular weight components. Preferably theprocess of interaction switching on the biochemical process or cascadeof interest requires one first heterologous DNA sequence of the nucleargenome and/or expression product of the first heterologous DNA sequence,and one second heterologous DNA sequence of the transfection vectorand/or expression product of the second heterologous DNA sequence.

The DNA sequences used according to the invention may be obtained viathe use of RNA sequences. Specifically, the DNA sequences of steps (a)or (b) may be an expression product of RNA sequences, e.g. of an RNAvirus.

In the absence of any one of the first and second heterologous DNAsequences or expression products of the first and second heterologousDNA sequences required for the process of interaction, none of thepresent first and second heterologous DNA sequences, expression productsof the first and second heterologous DNA sequences or the externallyadded low molecular weight components are able, alone or in combination,to switch on the biochemical process or cascade of interest. Moreover,the biochemical process or cascade of interest is not a process whichhas been silenced by a mechanism such as post-transcriptional genesilencing which may be still operating at a low level. The biochemicalprocess or biochemical cascade of interest is not operable in thetransgenic plant prior to the infection with a corresponding viraltransfection vector and prior to the optional addition of a lowmolecular weight component. Moreover, a viral transfection vectoraccording to the invention is unable to switch on the biochemicalprocess or biochemical cascade of interest in a plant not having thecorresponding first heterologous DNA sequence required according to theinvention. Finally, the biochemical process or cascade of interestcannot be switched on in a plant by contacting the plant with a lowmolecular weight component in the absence of the first and secondheterologous DNA sequences or expression products required for switchingon the process or cascade of interest according to the invention.

The process of the invention provides control over a biochemical processor cascade of interest with a hitherto unattainable technical precisionand environmental safety. Thereby novel applications in plantbiotechnology are available for solving problems which cannot be solvedby conventional technologies involving basal transgene expressionactivity in the plant, particularly when producing toxic substances orbiodegradable polymers.

Moreover, the precise control according to the invention allows to growa transgenic plant to a desired stage where the plant is best suited forperforming the biochemical process or cascade of interest withoutburdening the plant with a basal expression activity slowing down thegrowth of the plant. Once the plant is ready for efficiently performingthe biochemical process or cascade of interest, the process or cascadeof interest may be switched on and performed with high efficiency.Accordingly, the process of the invention allows to safely decouple thegrowth phase and the production phase of a transgenic plant.

Moreover, it is possible to design multi-component systems for multiplebiochemical processes or cascades of interest, whereby one or moredesired processes or cascades can be selectively switched on.

In a first embodiment, the system comprises a transgenic plantcontaining a heterologous DNA sequence providing an expression productwhich is necessary to control expression of a desired product encoded bya viral transfection vector. The system further comprises differentviral vectors each encoding a different product to be expressed in thetransgenic plant. Thereby, it is possible to safely use the sametransgenic plant for the production of different products depending onthe viral vector used. The advantage of this system is clear in thelight of the fact that it may take years to provide a stably transformedtransgenic plant whereas the preparation of a viral vector may beaccomplished in a few weeks.

In a second embodiment, the transgenic plant contains multipleheterologous DNA sequences which may encode for different desired geneproducts. Each of the multiple heterologous DNA sequences may becontrolled by a different viral vector. Thereby, it is possible toselectively control the heterologous DNA sequences of the transgenicplant by the choice of the corresponding viral vector.

Moreover, in a third embodiment, it is possible to design a systemwherein the process of interaction switching on the biochemical processor cascade of interest requires the infection with more than one viralvector and the optional application of one or more externally added lowmolecular weight components whereby present technology is even safer tooperate.

A biochemical process or cascade to be controlled according to theinvention may be any process or cascade which may take place in a livingplant system. Preferred biochemical processes or cascades lead to theproduction of a product in the plant. Examples of products of interestwhich may be obtained by the process of this invention includepolypeptides or proteins as primary products, (posttranslationally)modified or otherwise processed proteins which may be enzymes, proteinshaving a desired glycosylation pattern, non-proteinaceous low-molecularweight products and oligomerisation products thereof like carbohydratesor biodegradable plastics etc. Most preferred are pharmaceuticalpolypeptides.

Said biochemical process or cascade, notably expression of a protein,may involve formation of sub-genomic RNA, notably from a viraltransfection vector.

The process of controlling a biochemical process or cascade involves atleast the following two components:

-   -   (1) a transgenic plant containing a first heterologous DNA        sequence preferably not originating from a plant virus and    -   (2) a viral transfection vector containing a second heterologous        DNA sequence.

For the purposes of this invention, a heterologous DNA sequence is asequence which neither occurs naturally in the plant species employednor in the wild-type virus on which the viral vector is based on,respectively. Nevertheless, such a sequence may comprise sequenceportions native to the plant and/or the virus of interest besidesheterologous portions. Said heterologous DNA sequence may comprise morethan one functional element. Examples of such functional elementsinclude promoter, enhancer, transcription termination region, codingregion, non-translated spacer region, translation initiation region,IRES (internal ribosome entry site) region, stop codon etc. or partsthereof.

Said first heterologous DNA sequence is preferably heterologous to saidhost plant. Said first heterologous DNA sequence may be of viral origin.Preferably, however, said first heterologous DNA sequence is ofnon-plant viral origin, i.e it is not of plant viral origin. Said secondheterologous DNA (or RNA) sequence is preferably heterologous to thevirus on which the viral vector is based on. Said second heterologousDNA sequence may be of plant origin.

Infection of the transgenic plant (1) with a viral vector (2) triggers aprocess of interaction between said at least first and secondheterologous DNA sequence or expression product(s) thereof, thusswitching on the biochemical process or cascade of interest. The factthat the at least two components (1) and (2) are required means thatinteraction of said components is a necessary condition for switching onsaid biochemical process or cascade. Prior to said interaction, saidbiochemical process is not operable whereby “leaky” expression of atransgene cannot occur. In prior art systems, expression of a transgenecan merely be induced by a quantitative increase or an enhancement of analready existing, albeit lower, expression level. The present inventionnot only provides a quantitative increase but also a qualitative changein that a previously not operable process or cascade becomes operable.This advantage of the present invention is of particular importance whena biochemical process or cascades of interest involves formation of atoxic or growth-retarding product. According to the invention it ispossible to entirely separate plant growth and production of saidproduct whereby interference with or retardation of plant growth by thepresence of the desired product in the growing plant is avoided.Therefore, the stages of biomass accumulation and production of aproduct of interest may be decoupled.

The transgenic plant and the transgenic vector of the invention are notfunctional for controlling a biochemical process or biochemical cascadewith viruses or plants not containing the corresponding heterologous DNAsequences, respectively. Consequently, this invention represents asignificant progress in terms of biological safety in plantbiotechnology.

Said processes of interaction which are triggered by infecting thetransgenic plant with a viral vector and which lead to switching on of abiochemical process include DNA recombination, DNA replication,transcription, restriction, ligation, hybridisation, RNA replication,reverse transcription, RNA processing, splicing, translation, proteinfolding, assembly, targeting, posttranslational processing, enzymaticactivity. Said expression products of said first or said secondheterolgous DNA sequence include RNA, notably mRNA, and polypeptides orproteins.

Said process of interaction between said first and said secondheterologous sequences (and optionally further sequences) doespreferably not include complementation (genetic reassembly) of viralfunctions or of an infectious viral vector.

This invention preferably relates to multicellular plants. Examples forplant species of interest are monocotyledonous plants like wheat, maize,rice, barley, oats, millet and the like or dicotyledonous plants likerape seed, canola, sugar beet, soybean, peas, alfalfa, cotton,sunflower, potato, tomato, tobacco and the like. The fact that there arespecific viruses for each of such plants, contributes to the broadversatility and applicability of this invention. The viral transfectionvector used in this invention may be derived from any such plantspecific virus. The viral vector may be based on an RNA or on a DNAdouble-stranded or single-stranded virus. Specific examples of viraltransfection vectors are given below and in ANNEX A and ANNEX B.

In step (a), the plant may be a natural plant or a genetically modifiedplant. The genetic modification may be either in the nuclear genome ofthe plant or in an organelle genome such as plastid or mitochondriagenome. In step (a) a heterologous sequence is introduced in the nucleargenome, and preferably a stable genome modification is provided. Step(a) may be carried out more than once in order to introduce more thanone heterologous DNA sequence. In this way several heterologousfunctions may be introduced in the target plant e.g. for engineering awhole biochemical pathway.

In step (b), the transgenic plant obtained according to step (a) isinfected with a viral transfection vector. The infecting may be achievedby supplying the plant with an assembled virus particle, or withinfectious viral nucleic acids, or by activating a transfection processby release of viral nucleic acids previously incorporated into the plantgenome. The assembled virus particle may contain RNA and the infectiousviral nucleic acids may be RNA, notably if they are based on an RNAvirus (cf. examples 2 and 3).

More than one vector may be used to control the biochemical process orbiochemical cascade of interest. Preferably, only one vector containingthe desired heterologous sequence(s) is used for reasons ofreproducibility of the process. Infection may be done by contacting theviral vector with said transgenic plant. Preferably, mechanicalstimulation like rubbing or scatching of leaves or other plant tissuemay be used to initiate infection. Infection may also be achieved byactivating the viral vector previously integrated in the genome of thehost plant. Viral vectors capable of systemic infection of the plant arepreferred.

The infection of the plant in step (b) may further compriseAgrobacterium-mediated transfer of nucleic acid sequences into cells ofsaid plant. Agrobacterium-mediated transfer may e.g. be used tointegrate sequences into the genome of the host plant. A viral vectormay be activated from such sequences integrated the genome of the plant.An RNA virus-based vector may e.g. be activated by transcribing a cDNAcopy of said virus, notably by transcribing a cDNA copy integrated intothe genome. However, integration of sequences introduced into plantcells by Agrobacterium-mediated transfer do not have to lead tointegration into the genome. Agrobacterium-mediated transfer may providetransient expression of a gene flanked by T-DNA. Notably, sequences on aTi-plasmid may exert a function in the process of the invention withoutor before integration into the genome. If more than one vector isintroduced in step (b), the same or different methods may be used forthese vectors. Notably, more than one vector may be introduced byAgrobacterium-mediated transfer using different Agrobacterium strainssimultaneously (e.g. using an Agrobacterium mixture) or consecutively.

In one embodiment of the invention, a further vector in addition to saidviral infection vector may be introduced in step (b) of the process ofthe invention. Said further vector may be or may not be a viraltransfection vector. Said further vector may provide a further nucleicacid sequence as a necessary condition for switching on said biochemicalprocess of the invention (cf. example 6).

In another embodiment of the invention, infecting the plant in step (b)is achieved by introducing one or more vectors into cells of said plant,whereby said vector(s) are adapted to undergo processing to generate aviral transfection vector in cells of said plant. Three, four or morevectors may be introduced in cells of said plant in this embodiment.Preferably, two vectors are introduced. Said vectors may or may not beviral transfection vectors. Preferably, at least one of said vectors isa viral transfection vector (cf. example 6). However, according to thisembodiment, a viral transfection vector may also be generated fromintroduced vectors none of which is a viral transfection vector. Saidbiochemical process or pathway may be switched on by the assembly andappearance of said viral transfection vector in cells of the plant bysaid processing, triggered by measures (a) and (b) of the process of theinvention.

Steps (a) and (b) of the process of this invention may be carried out onthe same plant. However, it is preferred that a stable plant line isobtained according to conventional processes based on a plant in whichat least one first heterologous DNA sequence of interest was introducedaccording to step (a). Transgenic plants may then be grown from seeds ofa stably transformed plant, and infection according to step (b) may beperformed when initiation of said biochemical process is desired. Step(b) is preferably carried out in a greenhouse.

A viral transfection vector is a nucleic acid (RNA or DNA) ornucleoprotein which upon invading a wild type or genetically engineeredhost is capable of replication or amplification in cells of said hostand of amplification and/or expression of heterologous sequence(s) ofinterest. Preferably, said viral transfection vector is further capableof cell to cell movement. More preferably, a viral vector retainsadditional viral capabilities such as long distance movement, assemblyof viral particles or infectivity. In the process of this invention, aviral vector might not have all the properties mentioned above, but suchfunctions can be provided in trans in the context of host cell.Preferred viral transfection vectors encode and express a movementprotein. Further, they may encode a virus-specific DNA or RNA polymerase(replicase); a RNA-dependent RNA polymerase (RdRp) is preferred.

In a first specific embodiment of this invention, the biochemicalprocess of interest is expression of a heterologous DNA sequence ofinterest. This process may be called primary biochemical process. Thisprimary process results in an RNA or polypeptide molecule. In thesimplest case, one of the RNA or polypeptide molecule is the product ofinterest. In a biochemical cascade, the product of such a primaryprocess may cause a secondary biochemical process e.g. by way of itscatalytic activity or by way of regulating the other biochemicalprocess. In a biochemical cascade, more than one biochemical processtakes place, whereby each such process depends on a previous biochemicalprocess. Said controlling or switching is preferably directed to saidprimary biochemical process in this embodiment.

In the first specific embodiment, the heterologous DNA sequence ofinterest which is to be expressed, may either be a first heterologousDNA sequence of the plant nuclear genome or a second heterologous DNA(or RNA) sequence of said viral transfection vector.

In a second specific embodiment of this invention, the biochemicalprocess of interest is the production of non-proteinaceous compound ofinterest by the plant.

In a further specific embodiment of the invention, a process ofcontrolling a biochemical process (II) or biochemical cascade (III) ofinterest in a plant is provided, said process being characterized bycomprising the following steps:

-   -   (a) introducing into the nuclear genome of the plant one or more        first heterologous nucleic acid sequences,    -   (b) infecting the plant with at least one vector containing in        its genome one or more second heterologous nucleic acid        sequences,        thus triggering a process of interaction (I) in the plant        between    -   (i) one or more first heterologous nucleic acid sequences of the        nuclear genome and/or expression products of the first        heterologous nucleic acid sequences, and    -   (ii) one or more second heterologous nucleic acid sequences of        the transfection vector and/or expression products of the second        heterologous nucleic acid sequences, and    -   (iii) optionally one or more externally added low molecular        weight components, whereby a viral transfection vector is        generated in cells of said plant, thus switching on the        biochemical process (II) or biochemical cascade (III) of        interest that was not operable prior to said interaction.        Further, specific embodiments of this process may be as        described above, where applicable.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A is a schematic representation of a process according to theinvention.

FIG. 1B is a schematic representation of possible classes of processesof interaction in an infected plant cell.

FIG. 2 depicts crTMV-based vectors pIC1111 and pIC1123 containingIRES_(cp,148) ^(CR)-Ac transposase and and IRES_(mp,75) ^(CR)-Actransposase, respectively. Also shown is the T-DNA region of binaryvector pSLJ744 containing p35S::Ds::GUS-3′ocs.

FIG. 3 depicts crTMV-based vectors pIC2541 and pIC2531 containingIRES_(cp,148) ^(CR)-Cre recombinase and and IRES_(mp,75) ^(CR)-Crerecombinase, respectively. Also shown is the T-DNA region of the binaryvector pIC2561 containing the GUS gene flanked by two loxP sites indirect orientation.

FIG. 4 depicts crTMV-based vectors pIC2541 and pIC2531 (see also FIG. 3)in combination with the T-DNA region of the binary vector pIC1641containing the GUS gene flanked by two inverted loxP sites.

FIG. 5 depicts the T-DNA region of the binary vector pIC2691 carryingthe GUS gene under control of T7 promoter and crTMV-based vector pIC2631containing the T7 polymerase gene.

FIG. 6 shows X-gluc stained leaves of transgenic Arabidopsis plantstransformed with pSLJ744. Transcription of the GUS gene is prevented bythe insertion of Ds element.

A—leaves inoculated with the transcript from pIC1123.

B—leaves inoculated with the transcript from pIC1111.

C—leaves inoculated with water.

FIG. 7 depicts the TMV-based viral provectors pICH4371 and pICH4461 endof provector (RdRp: RNA dependent RNA polymerase; MP: movement protein;sGFP: synthetic green fluorescent protein; 3′NTR: 3′non-translatedregion of TMV; sgp: subgenomic promoter).

FIG. 8 depicts the T-DNA of binary vector pICH1754 providing a Crerecombinase expression cassette.

FIG. 9 depicts a scheme of formation of viral vectors from provectors inthe presence of Cre recombinase.

Appendices 1 to 11 depict vectors and constructs used in example 6.DETAILED DESCRIPTION OF THE INVENTION

As shown by FIG. 1A, the present invention provides a process ofcontrolling a biochemical process or biochemical cascade of interest ina plant whereby the process comprises a process of interaction (I),switching on a biochemical process of interest (II), which in turn maybe causal for a biochemical cascade of interest (III). The process ofinteraction may involve any one of the following reactions orcombinations of the reactions of DNA, RNA and Proteins. DNA reactionscontemplated in this invention are restriction, recombination,replication, transposition, amplification, and transcription. RNAreactions contemplated in this invention are RNA processing,replication, reverse transcription, hybridisation, and translation.Protein reactions contemplated in this invention are protein processing,folding, assembly, post-translational modifications, activation,targeting, binding activity modification, signal transduction. Process(III) may be present or absent. The production of a product is thepreferred result of the process of the invention.

As shown by FIG. 1B, possible processes of interaction may belong to oneor more of the classes of interaction shown by the figure. In thefigure, a transgenic plant cell infected with a viral transfectionvector is shown schematically. The recombinant plant genome contains oneor more heterologous DNA sequences which may lead to one or moreexpression products. The genome of the viral transfection vectorcontains one or more heterologous DNA sequences which may lead to one ormore expression products. The transgenic plant cell may be contactedwith one or more low molecular weight components capable of entering thecell. In the process of interaction switching on the biochemical processor biochemical cascade of interest in the plant cell, the followinginteractions may occur which are indicated by arrows in FIG. 1B. One ormore heterologous DNA sequences of the recombinant plant genome mayinteract with one or more heterologous DNA sequences of the viraltransfection vector. One or more heterologous DNA sequences of therecombinant plant genome may interact with one or more expressionproducts of the heterologous DNA sequences of the viral transfectionvector. One or more expression products of the heterologous DNAsequences of the recombinant plant genome may interact with one or moreexpression products of the heterologous DNA sequences of the viraltransfection vector. The expression product may be an RNA or apolypeptide. Any of these interactions may also involve or require thepresence of one or more low molecular weight components added externallyto the infected transgenic plant cell. The low molecular weightcomponents may be necessary or desired for switching on or for promotingthe biochemical process or biochemical cascade of interest. The lowmolecular weight components are unable to switch on the processes orcascades of interest in the absence of the viral transfection vector orthe heterologous DNA sequence in the plant nuclear genome.

According to the first specific embodiment of this invention, a novelprocess to achieve transfection-based reliable control over either theexpression of a transgene stably integrated into a plant, or overexpression of a heterologous gene of a viral vector inside a transgenicplant host is provided. This process makes use of an interaction of atleast two heterologous DNA sequences or expression products thereof,which is triggered only when the virus vector infection process isinitiated. One of these sequences may be stably incorporated in theplant nuclear genome and the other one is provided by said viral vector.Such a switchable two-component expression system can be used to controla biochemical process or cascade that may be controlled at variouslevels, e.g. by triggering interaction reactions such as, but notlimited to: DNA recombination, replication, transcription, restriction,RNA replication, reverse transcription, processing, translation, proteinfolding, assembly, targeting, posttranslational processing, enzymaticactivity, etc.

This process requires at least a heterologous DNA in a transgenic plantand a recombinant virus-based vector comprising a heterologous DNA orRNA sequence. The general scheme of this process is shown in FIG. 1. Atransgenic plant containing in its nuclear genome one or more stablyintegrated heterologous DNA sequence(s) of interest can be engineeredusing standard transcriptional or translational vectors and standardtransformation protocols. Construction of transcriptional vectors forstable plant transformation has been described by numerous authors (forreview, see Hansen & Wright, 1999, Trends in Plant Science, 4, 226-231;Gelvin, S. B., 1998, Curr. Opin. Biotech., 9, 227-232). The basicprinciple of all these constructs is identical: a fully functionaltranscription unit consisting of, in 5′ to 3′ direction, aplant-specific promoter, the structural part of a gene of interest and atranscriptional terminator has to be introduced into the plant cell andstably integrated into the genome in order to achieve expression of thegene of interest. Construction of translational plant vectors isdescribed in German Patent Application Nos. 100 49 587.7 and 10061150.8(ANNEX A and ANNEX B). The principal difference to transcriptionalvectors is that translational vectors do not require a transcriptionalpromoter for expression of the gene of interest but rely on the planttranscription machinery following their integration into plant genome.

Different methods may be used for the delivery of an expression vectorinto plant cells such as direct introduction of said vector into thecells by the means of microprojectile bombardment, electroporation orPEG-mediated transformation of protoplasts. Agrobacterium-mediated planttransformation also represents an efficient way of vector delivery.Thus, DNA may be transformed into plant cells by various suitabletechnologies such as by a Ti-plasmid vector carried by Agrobacterium(U.S. Pat. No. 5,591,616; U.S. Pat. No. 4,940,838; U.S. Pat. No.5,464,763), particle or microprojectile bombardment (U.S. Pat. No.5,100,792; EP 00444882B1; EP 00434616B1). Agrobacterium can serve notonly for stable nuclear transformation, but also for an efficientdelivery of T-DNA for transient expression of gene(s) of interest. Thisso called agroinfiltration protocol was first developed to analyzeforeign genes expression and gene silencing in plants (Kaplia et al.,1997, Plant Science, 122, 101-108; Schöb et al., 1997, Mol. Gen. Genet,256, 581-588).

In principle, other plant transformation methods can also be used e.g.microinjection (WO 09209696; WO 09400583A1; EP 175966B1),electroporation (EP00564595B1; EP00290395B1; WO 08706614A1) etc. Thechoice of the transformation method depends on the plant species to betransformed. For example, microprojectile bombardment may be preferredfor monocots transformation, while for dicots, Agrobacterium-mediatedtransformation gives generally better results.

Construction of plant viruses for the expression of non-viral genes inplants has been described in several papers (Dawson et al., 1989,Virology, 172, 285-293; Brisson et al., 1986, Methods in Enzymology,118, 659; MacFarlane & Popovich, 2000, Virology, 267, 29-35; Gopinath etal., 2000, Virology, 267, 159-173; Voinnet et al., 1999, Proc. Natl.Acad. Sci. USA, 96, 14147-14152) and can be easily performed by thoseskilled in the art.

In one specific embodiment of our invention, the transgene in a plantgenome is separated from its promoter by a DNA insert sufficiently longto prevent the transcription of said transgene (FIGS. 2, 3). Said DNAinsert may be, for example, a non-autonomous transposable element or anyDNA fragment flanked by unidirected sites recognizable by asite-specific DNA recombinase. The appropriate transposase orsite-specific DNA recombinase may be delivered by a viral vector whichfunctions as a vector switch (FIGS. 2, 3). After expression of saidvector-encoded transposase or recombinase, the catalytic activity ofsuch an enzyme leads to excision of the DNA insert or fragment that wasseparating the promoter from the transgene switching on expression ofthe transgene (FIG. 6).

Site-specific recombinases/integrases from bacteriophages and yeasts arewidely used for manipulating DNA in vitro and in plants. Preferredrecombinases-recombination sites for the use in this invention are thefollowing: Cre recombinase-LoxP recombination site, FLP recombinase-FRTrecombination sites, R recombinase-RS recombination sites, etc.Transposons are widely used for the discovery of gene function inplants. Preferred transposon systems for use in the present inventioninclude Ac/Ds, En/Spm, transposons belonging to “mariner” family, etc.

In another embodiment of this invention, the transgenic plant may carrya promoterless transgenic sequence flanked by two inverted loxP sites(FIG. 4). Such an orientation of recombination sites may lead to theinversion of the flanked DNA sequence when exposed to Cre recombinase.As a consequence of such an inversion, a promoterless gene withnosterminator will be placed from anti-sense in sense orientation towardsthe constitutive promoter. Example 4 exemplifies this approach using apromoterless GUS gene with a nos terminator.

Another embodiment of this invention describes the possibility toassemble a functional viral vector construct in vivo in an engineeredplant cell. This means required elements of a viral vector (precursors)are delivered separately with two (FIG. 7) or more constructs into theplant cell. After e.g. Agrobacterium tumefaciens mediated delivery ofsuch precursors into a plant cell expressing the site-specific DNArecombinase (FIG. 8), site specific recombination can lead to theassembly of functional viral vector expressing transgene of interest(example 6, FIG. 9).

Heterologous transcription factors and RNA polymerases may also be usedas transgene switches. This approach is demonstrated in Example 5wherein a transgenic plant carries the GUS gene under control of abacteriophage T7 promoter (see FIG. 5). No GUS expression can bedetected in transgenic Arabidopsis containing such construct as theplant RNA polymerases do not recognize prokaryotic promoters. Viraldelivery of the bacteriophage T7 RNA polymerase triggers expression ofthe GUS gene (FIG. 5).

The expression of a plant transgene that is under control of abacteriophage promoter (e.g. T3, T7, SP6, K11) with the correspondingDNA/RNA polymerase delivered by a viral vector may be another efficientapproach for the development of transgene switches contemplated in thisinvention. Another useful approach may be the use of heterologous orchimaeric or other artificial promoters which require heterologous orengineered transcription factors for their activation. In some cases,the existing inducible systems for transgene expression may be used.Examples are the copper-controllable (Mett et al., 1993, Proc. Natl.Acad. Sci. USA., 90, 4567-4571) and the ethanol-inducible geneexpression systems (Caddick et al., 1998, Nature Biotech., 16, 177-180)which may be modified such that the transcription factors (ACE1 forcopper-inducible or ALCR for the ethanol-inducible system) are providedin trans by viral delivery, thus further reducing the leakiness of theexpression systems. Alternatively, heterologous transcription factorsmay be modified in such that no activating ligand-inducer will berequired to drive the transcription factor into the active state.

Other embodiments contemplated in this invention include triggeringreactions such as DNA restriction and/or DNA replication. An example ofa biochemical cascade that can be triggered by restriction is atwo-component system wherein a DNA sequence containing an origin ofreplication and being integrated into a nuclear genome is specificallyexcised and converted into an autosomally replicating plasmid by arare-cutting restriction enzyme delivered by viral vector, thustriggering the cascade. Alternatively, a DNA viral vector with amodified system of replication initiation may be made operable only inthe presence of a factor in a transgenic host that allows for efficientreplication of the modified viral vector in question.

There are numerous reactions affecting RNA molecules that may be used asefficient triggering devices of a cascade according to the presentinvention. These include, inter alia, reactions such as RNA replication,reverse transcription, editing, silencing, or translation. For example,a DNA derived from an viral RNA vector may be reverse transcribed by atransgenic host into a DNA which in turn could participate in processessuch as DNA integration into a nuclear genome or DNA-mediatedmutagenesis.

Another recombinant viral switch contemplated under the invention is aprocess that relies on posttranslational modification of one or moretransgene expression products. There are many possible implementationsof such switches that could operate by controlling steps such aspolypeptide folding, oligomer formation, removal of targeting signals,conversion of a pro-enzyme into an enzyme, blocking enzymatic activity,etc. For example, expression of a polypeptide from a viral expressionvector may trigger a biochemical process of interest only if agenetically engineered host specifically cleaves a pro-enzyme thusconverting it into an active enzyme, if a product is targeted to aparticular cellular compartment because of the host's ability to cleaveor modify targeting motif, or if a product is specifically mobilized dueto the removal of a specific binding sequence.

The process of this invention relies on the interaction of at least twocomponents, but multi-component systems based on interactions betweenmore than one heterologous DNA in host nuclear genome or more than oneviral transfection vectors are also contemplated. The same is true withregard to multi-component systems that involve, in addition to the abovenamed two components (heterologous DNA or its product in a host plantand a heterologous DNA or its product in a viral vector), additionalelements such as low molecular weight effectors or nucleic acids orproteins that are not integrated into a plant chromosome. Such a lowmolecular weight component is defined as a non-proteinaceous molecule orion having a molecular weight of less than 5 kD. The ultimate purpose ofa recombinant switch system contemplated herein is an operationalcontrol of a process in a plant production system, such as biochemicalpathway or a cascade of biochemical reactions of interest. A pathway ora biochemical cascade is a chain of biochemical reactions in a hostproduction system that upon completion, yields a specific product,effect or trait.

The approaches described herein, in addition to being versatile andleakage-proof gene switches, provide an efficient production controlmethod. The two-component process described above is in essence a“key-lock” system, whereby a company can efficiently control access toproduction by selling the transfection switch component.

EXAMPLES

With regard to additional disclosure of specific vectors and constructsused in the following examples, reference is made to ANNEX A and ANNEXB.

Example 1

Construction of Viral Vectors for Plant Infection, Carrying the GenesInvolved in DNA Recombination: Ac Transposase and Cre Recombinase

Series of crTMV-based viral expression vectors carrying the genesinvolved in DNA recombination, were constructed according to a standardmolecular biology protocols (Maniatis et al., 1982, Molecular cloning: aLaboratory Manual. Cold Spring Harbor Laboratory, New York). Detailedinformation concerning commonly used vectors, genes and gene fragmentsused in this and the following examples can be found in public domaindatabases. Two-step cloning strategy was used for all constructs. First,an intermediate construct was made to fuse the gene of interest (GUS)with the appropriate IRES-sequence and the 3′-nontranslated region (NTR)of the crTMV (pseudoknots and t-RNA-like structure). For theIRESmp75^(CR) and IREScp148^(CR)-fusions (Skulachev et al. 1999,Virology 263, 139-154) the gene of interest (GUS) was subcloned into theplasmid pIC766 (IRESmp75^(CR)-GUS-3′-NTR in pBS(SK+) and into theplasmid pIC751 (IREScp148^(CR)-GUS-3′-NTR in pBS(SK+), respectively.Convenient restriction sites for sub-cloning, like Nco I at the 5′-endand BamH I- or Xba I at the 3′-end of the gene of interest wereintroduced by PCR if necessary. DNA sequencing analysis was used toconfirm the sequences of all PCR-amplified parts of the construct.

In the final step of cloning, the IRES/GUS/3′-NTR-fragment wassub-cloned further into the viral expression vector pIC797 (T7promoter—crTMV cDNA with the GUS gene following the viral CP gene(RdRp-MP-CP-HindIII-IRESmp228^(CR)-GUS-3′NTR)-NotI-Xbal-Spel-BamHI inpUC19) as a HindIII/NotI fragment. For this purpose, the plasmid pIC797was digested with SacII and NotI, the large fragment was gel purifiedand ligated with the 1.3 kb SacII/HindIII fragment of the same plasmidand the HindIII/NotI-fragment of the intermediate construct (pIC2251 forCre recombinase). In case of the Ac-transposase a four-fragmentsligation was necessary due to the presence of a HindIII-restriction sitein the coding part of the Ac gene. The final constructs (pIC1111 andpIC1123 for Ac transposase; pIC2541 and pIC2531 for Cre recombinase) areshown in FIGS. 2 and 4 respectively.

Example 2

In Vitro Transcription of Viral Vector Constructs

The plasmids pIC1111, pIC1123, pIC2541 and pIC2531 (FIGS. 2 and 4,respectively) were linearized by digestion with Not I restrictionendonuclease. The linearized plasmids were transcribed in vitro asdescribed by Dawson et al. (1986, Proc. Natl. Acad. Sci. USA., 83,1832-1836). Quality and quantity of full-length RNA transcripts weredetermined by agarose gel electrophoresis (Maniatis et al., 1982,Molecular cloning: a Laboratory Manual, Cold Spring Harbor Laboratory,New York).

Example 3

Activation of a Transgene Stably Integrated in a Plant Genome byVirus-Delivered Ac Transposase

The T-DNA of plasmid pSLJ744 (obtained from J. Jones, SainsburyLaboratory, JIC, Norwich, UK) (FIG. 2) was introduced in Arabidopsisthaliana (Col-0) plants as descried by Bent et al., (1994, Science, 285,1856-1860). Seeds were harvested three weeks after vacuum-infiltration,sterilised and screened for transformants on GM+1% glucose medium(Valvekens et al., 1988, Proc. Natl. Acad. Sci. USA, 85, 5536-5540.)containing 50 mg/L kanamycin. Rosette leaves of five weeks oldArabidopsis transformants were inoculated with full-lengthtranscript-RNA as obtained in example 2 by mechanical wounding. For thispurpose, the RNA was mixed with 3× GKP-buffer (50 mM glycine, 30 mMK₂HPO₄, 3% celite, 3% benthonite) and scratched gently on the upper sideof the leaves. The T-DNA of plasmid SLJ744 contained a non-autonomous Dselement inserted between the CaMV 35S promoter and the GUS gene (FIG.2). Excision of the Ds element caused by action of virus-delivered Actransposase leads to the expression of the GUS-gene, which can be easilymonitored by histochemical staining of inoculated leaves (Jefferson,1987, Plant Mol. Biol. Reporter, 5, 387-405). Inoculated leaves werecollected 9-14 days after the transfection with full-length transcriptRNA. Samples were infiltrated using X-gluc solution (Jefferson, 1987,Plant Mol. Biol. Reporter, 5, 387-405). After incubation overnight at37° C., the leaves were destained in 70% ethanol and examined by lightmicroscopy. Large sectors of GUS-stained tissues were observed inprimarily inoculated leaves. No GUS staining was detected in the controltransgenic plants inoculated by distilled H₂O. The results are shown inFIG. 6. The sectors of GUS staining are consistent with the sectors ofviral infection in primarily inoculated leaves. This is evidence for thehigh efficiency of this approach: Ds excision and, as a consequence, GUSexpression took place in all infected cells. In comparison, constantpresence of Ac transposase in plants carrying a copy of the Ac transgenestably integrated in the genome leads to Ds excision sectors only in aminor fraction of the plant tissue (results not shown).

Example 4

Activation of a Transgene Stably Integrated in the Plant Genome byVirus-Delivered Cre Recombinase

Two different constructs pIC2561 and pIC1641 (FIGS. 3 and 4,respectively) with loxP-recombination sites were designed as targets forCre-mediated recombination. In construct pIC2561, the GUS gene with the3′NOS transcription termination signal is flanked by two directloxP-sites. This fragment was inserted between the CaMV 35S promoter anda synthetic GFP gene (sGFP). The recombination between the two loxPsites, once exposed to virus-delivered Cre recombinase, leads toexcision of the GUS gene. This event can be easily monitored by GFPexpression and absence of GUS-acitivity in the inoculated leaves.

For the construction of plasmid pIC2561, the SLJ4K1 (Jones et al. 1992,Transgenic Research 1, 285-292) the GUS gene was amplified with primerscarrying loxP sites and Cla1 (5′CCG ATC GAT ATA ACT TCG TAT AGC ATA CATTAT ACG AAG TTA TAT GTT ACG TCC TGT AGA AAC CC3′) and Nco1 (5′GGC CATGGA TAA CTT CGT ATA ATG TAT GCT ATA CGA AGT TAT TGC ATG CCT GCA GGT CGATCT AGT AAC3′) restriction sites were introduced at the 5′ and 3′ endsof the gene, respectively. Said PCR-product was digested with Cla1 andNco1 restriction enzymes and subcloned into the Nco1-Cla1 sites of theplasmid pIC591 (pHBT:ClaI/NcoI-sGFP-3′NOS). The HBT promoter (Sheen, J.1995, EMBO J., 12, 3497-3505) (not functional in Arabidopsis) of thisintermediate construct was replaced by the CaMV 35S promoter by ligatingtogether its gel-purified large HindIII/Klenow—Cla1 fragment with the1.4 kb EcoR1/Klenow—Cla1 fragment of (35S promoter) of SLJ4K1.Functional clones were determined in microprojectile co-bombardmentexperiments with DNA of pIC1422 (cre recombinase under control of HBTpromoter). For further subcloning into the binary vector pICBV1(proprietary development of Icon Genetics AG, Munich, Germany, however,any other binary vector is suitable as well), the pICBV1-DNA wasdigested with EcoRI and Ecl13611 restriction enzymes, gel-purified andligated with the large Xho1/Klenow—EcoR1 fragment(p35S:-loxP-GUS-3′OCS-loxP-sGFP-3′NOS) of said functional intermediateclone. The T-DNA region of the final construct pIC2561 is shown in FIG.3.

The second construct carrying the GUS gene flanked by two inverted loxPsites is shown in FIG. 4. To make this construct, two PCR primers (5′CTGAAG CTT ATA ACT TCG TAT AGC ATA CAT TAT ACG AAG TTA TAC CAT GG CTG CAGATA ACT TCG TAT3′ and 5′GCC TCG AGA TAA CTT CGT ATA ATG TAT GCT ATA CGAAG TT ATC TGC AGC CAT GGT ATA ACT TCG TA3′) with 18 bb of complementary3′ ends were designed, annealed and filled in with the Klenow fragmentof DNA polymerase I.

The final DNA fragment contained two inverted loxP sites separated byPst1, Nco1 and flanked by Xho1 (from the Pst1 side) and Hind1111restriction sites. After a Xho1-Hind111 digestion, the fragment wasligated with large Xho1-Hind111 fragment of pSLJ4D4 (Jones et al., 1992,Transgenic Research, 1, 285-292). The resulting plasmid was digestedwith Pst1-Nco1, gel-purified and ligated with the 2.6 kb Nco1-Pst1fragment of pSLJ4D4.

As the final step of cloning, the whole cassette (CaMVp35S-loxP-3′nos-GUS-loxP) was subcloned into the binary vector pBIN19(Bevan, M. 1984, Nucl. Acid Research, 12, 8711-8721) asHindIII/EcoRI-fragment. The T-DNA of this construct (pIC1641) is shownin FIG. 4. Transgenic Arabidopsis lines were obtained by Agrobacteriumtumefaciens mediated transformation according to the modified vacuuminfiltration protocol of Bent et al. (1994, Science, 265, 1856-1859).The presence of the transgene in segregating T1-population was confirmedby PCR-analysis.

Transcription of viral a cDNA clone (pIC2531) and inoculation oftransgenic Arabidopsis lines with viral RNA was performed as describedin examples 2 and 3, respectively.

GFP and GUS Detection

A LEICA stereo fluorescent microscope system was used to monitor GFPexpression (excitation at 450-490 nm, emission at 500-550 nm). The sGFPused in our experiments can be excited by blue and UV-light. GUSdetection was performed as described in example 3.

Example 5

Activation of a Transgene Stably Integrated in a Plant Genome byVirus-Delivered T7 RNA Polymerase

Construction of the Vectors

The binary vector for plant transformation with the GUS gene reporterunder control of the T7 promoter was made as follows. The gel-purified 2kb BssH11/T4 polymerase—Sal 1 fragment of pIC057 carrying the T7promoter-GUS gene construct was ligated with Sma1-Sal1 digestedexpression vector pIC056, adding the 35S transcription terminationsignal to the 3′ end of the GUS gene. The resulting construct pIC2641was digested with Sac1 and Xho1, gel-purified from the vector backboneand ligated with Sac1/Sal1 digested pBIN19. The final construct pIC2651(FIG. 5) was used for the Arabidopsis transformation as described above.The viral vector expressing T7 polymerase was made as follows. Theplasmid pIC2603 was digested with Sph1 and Sal1 and the gel purified 2.8kb fragment carrying the T7 polymerase gene was ligated with the largeNco1-Sal1 fragment of pIC1018. Resulting plasmid pIC2621 has the T7 geneflanked by the IRESmp75^(CR) at its 5′ end and by the 3′ nontranslatedregion (NTR) of crTMV at its 3′ end. The final cloning step included theligation of the small Hind111-Not1 fragment of pIC2621 with the largeSac11-Not1 and small Sac11-Hind111 fragments of pIC1087. The finalconstruct pIC 2631 (FIG. 5) containing the T7 polymerase gene in a crTMVviral vector was used for transcription and plant transfection asdescribed in examples 2 and 3, respectively.

Example 6

Activation of a Transgene from Viral Amplicon Precursors

Construction of the Vectors

In order to introduce LoxP-sites recognized by Cre recombinase into abasic construct, IPCR was performed with primers containing LoxP-sitesin opposite orientation flanked by convenient restriction sites (primer1: 5′-TATCTGCAGG AGCTCATAAC TTCGTATAAT GTATGCTATA CGAAGTTATA AGCTTCTGGCCGTCGTTTTA C-3′; primer 2: 5′-CTCCTGCAGA TAACTTCGTA TAATGTATGCTATACGAAGT TATCTCGAGG AATTCGGCGT AATCATGGTC A-3′). These primers wereannealed to the multi-cloning site of the pUC119 vector in order toamplify the whole plasmid in an IPCR-reaction. Overlapping sequences ofthe primers contained a Pst I-restriction site. After restriction of theIPCR-product with Pst I and religation, the intermediate constructpICH1212 (Appendix 1) was obtained.

The Xho1-EcoR1 fragment of MP-gene containing a translation stop codon25 AA before the natural translation termination signal was ligated withXho1-EcoR1 large fragment of PICH1212. In the resulting constructpICH3431 (Appendix 2) the 3′-MP-part is located next to a LoxP-site. Tofuse this MP-LoxP element to the 5′-part of a MP-gene in a vector, whichcontains also the Arabidopsis Actin 2-promoter and the RdRp-polymerase,the MP-LoxP element from pICH3431 was subcloned as EcoRI-Ecl136IIfragment into the plasmid pICH3301 (Appendix 3) cut with EcoRI and NotI,resulting in the plasmid pICH3461 (Appendix 4). The NotI restrictionsite was treated with Klenow fragment of DNA polymerase 1 beforesubcloning. The KpnI-XhoI and Xho-HindIII fragments at 5′-end of theresulting vector were further used for cloning into the Kpn1 andHindIII-treated binary vector pICBV10 (T-DNA region of pICBV10 is shownin Appendix 5) in a three fragment ligation reaction. The finalconstruct pICH4371 is depicted in FIG. 7.

For making a 3′-end of the viral vector precursor, an XhoI-NcoI fragmentcontaining a LoxP site next to an Ω-leader-sequence from constructpICH2744 (Appendix 6) was subcloned into plasmid pICH1721 (Appendix 7)to fuse the LoxP-site/Ω-leader-sequence-element to the 5′-end of thesGFP-gene which was flanked by a 3′NTR-sequence at the 3′-end (constructpICH3421, Appendix 8). In order to add a nopaline synthase transcriptiontermination signal to this ORF, plasmid pICH3421 was cut by KpnI andNotI and the resulting small fragment was cloned into the plasmidpICH3232 resulting in construct pICH3441 (Appendix 9). For theAgrobacterium tumefaciens-mediated delivery this 3′-end of the viralprecursor vector was further subcloned into the binary vector pICBV10(Appendix 5) as KpnI/HindIII fragment. The final construct pICH4461 isshown in FIG. 7.

The Cre recombinase construct pICH529 (wheat histone H4promoter-LoxP-Cre recombinase-NOS terminator, see Appendix 10) wasmodified to clone the Cre recombinase into the binary vector used forobtaining nuclear transformants of Nicotiana. First the wheat H4promoter was replaced by the Actin2 promoter from Arabidopsis bysubcloning an Ecl136II/Pst l-fragment from construct pIC04 (ArabidopsisActin2-promoter without intron, not shown) into pICH529 digested withHindIII (blunt) and Pstl. This resulted in construct pICH1262 (Appendix11). In order to replace the NOS-terminator by the OCS-terminatorflanked at its 5′-end by a LoxM recombination-site, the OCS-terminatorwas PCR-amplified from plasmid pICH495 (NOS promoter-BAR-gene-OCSterminator, not shown) and further subcloned as SphI/SacI-fragment intothe plasmid pICH1262 , producing the construct pICH1321 (not shown). Thesequence of the forward primer (5′-CGGCATGCAT AACTTCGTAT AATCTATACTATACGAAGTT AGGATCGATC CTAGAGTCCT GC-3′) used for this amplification,included the SphI-restriction site and the LoxM-recombination site. TheSacI-restriction site at the 3′-end of the PCR-product was introduced bythe sequence of the reverse primer (5′-CGGAGCTCGT CAAGGTTTGACCTGCACTTC-3′). Finally, the resulting construct (Actin2promoter-LoxP-Cre recombinase-LoxM-OCS terminator) was further subclonedinto the binary vector pIC00015 as NotI/SacI-fragment, resultinginconstruct pICH1754 (FIG. 8). To clone this fragment into the binaryvector it was necessary to fill in the NotI-site of the fragment and theEcoRI-site in the polylinker of the binary vector.Transformation oftobacco leaf discs

Transgenic Nicotiana lines (species tabacum and benthamiana), containingT-DNA of pICH1754, were obtained by Agrobacterium-mediatedtransformation of leaf discs as described by Horsch et al., (1985,Science, 227, 129-131). Leaf discs were incubated for 30 min withAgrobacterium strain GV3101 transformed with the construct pICH1754.After three days of incubation on medium (MS-medium 0.1 mg/l NAA, 1 mg/lBAP) without selective agent, selection of transformants was performedon the same MS-medium supplemented with 100 mg/L Kanamycin. In orderoreduce the growth of Agrobacterium, the medium was also supplementedwith 300 mg/L carbenicilin and 300 mg/L cefataxime. Regenerants wereincubated on selective MS-medium without hormones supplemented with thesame concentration of the selective agents to induce the rooting. Thepresence of the transgene in segregating T2-populations was confirmed byPCR-analysis.

Delivery of Viral Vector Precursors by Agro-Infiltration

The agroinfiltration of transgenic tobacco plants was performedaccording to a modified protocol described by Yang et al., 2000, PlantJournal, 22(6), 543-551. Agrobacterium tumefaciens strain GV3101transformed with individual constructs (pICH4371 and pICH4461) was grownin LB-medium supplemented with Rifampicin 50 mg/l, carbencilin 50 mg/land 100 μM acetosyringone at 28° C. Agrobacterium cells of an overnightculture (5 ml) were collected by centrifugation (10 min, 4500 g) andresuspended in 10 mM MES (pH 5.5) buffer supplemented with 10 mM MgSO₄and 100 μM acetosyringone. Bacterial suspension was adjusted to a finalOD₆₀₀ of 0.8. In case of delivery of several constructs agrobacterialclones carrying different constructs were mixed before infiltration.

Agroinfiltration was conducted on near fully expanded leaves that werestill attached to the intact plant. Bacterial suspension was infiltratedwith a 5 ml syringe. By infiltrating 100 μl of bacterial suspension intoeach spot (typically 3-4 cm² of infiltrated area) eight to 16 spotsseparated by veins could be placed in a single tobacco leaf. Afterinfiltration plants were further grown under greenhouse conditions at22° C. and 16 h light.

Sixteen days after infiltration, leaves of transgenic tobacco plants(pICH1754, Nicotiana tabacum) infiltrated with construct pICH4371 andpICH4461 showed growing sectors of strong GFP-expression which could beobserved under UV-light on intact plants. No GFP-expression was visibleon leaves of non-transformed tobacco infiltrated with the sameAgrobacterium suspension mix.

Annex A Vector System for Plants FIELD OF INVENTION

This invention relates to a vector capable of amplification andexpression and/or suppression of a gene in a plant, as well as usesthereof, and a method and pro-vector for generating said vector.

BACKGROUND OF THE INVENTION

Vectors for genetic engineering of plants are highly desirable for theproduction of proteins, for endowing a host plant with a new trait, forsuppressing a gene of the host plant, or for determining the function ofa gene, notably a gene determined by genomics. Vectors, notably viralvectors, for the genetic engineering of plants are already known. Thesemust be capable of infection, amplification and movement (bothcell-to-cell and long-distance) in a plant in addition to having atleast one sequence for gene expression or suppression. Prior art vectorsrely on subgenomic promoters as transcriptional elements. A subgenomicpromoter has the effect that, in a transfected plant cell, transcriptionof a vector nucleic acid sequence starts in part at said subgenomicpromoter to generate a shorter RNA so that translation of a genedownstream from said promoters by the plant translation machinery isenabled. Translation may then proceed cap-dependent. Such multipletranscriptions are kinetically disadvantageous because of waste ofreplicase capacity.

Such vectors have a number of further shortcomings. The introduction ofa virus subgenomic promoter into a vector sequence makes said sequencelonger and thus less efficient. Moreover, the presence of severalidentical or similar subgenomic promoters which are well adapted totranscription in the host gives rise to frequent recombination eventsand instability with loss of sequence portions. On the other hand, ifsignificantly different subgenomic promoters are used, recombination maybe suppressed but such promoters may be too different to be effectivelyrecognized by the transcription system, which means loss of efficiency.Moreover, vectors are usually highly integrated entities with severalinterdependent functional elements or genes tightly packed into asequence. This is the reason why the operability of a vector for certainheterologous genes or the like is somewhat idiosyncratic and frequentlygives unpredictable results, notably in terms of infectivity andexpression. Further, the available sequence space for promoters isusually constrained if sequence overlaps with upstream genes arepresent.

Therefore, it is an object of this invention to provide a novel vectorfor plant genetic engineering which is capable of efficient and stableoperation in a host plant. It is a further object to provide a vectorwhich is capable of high-level expression of a gene in a plant.

It has been surprisingly found that these objects can be achieved with avector capable of amplification and expression of a gene in a plantcomprising a nucleic acid having a sequence for at least one non-viralgene to be expressed and having or coding for at least one IRES elementnecessary for translation of a gene downstream thereof.

It has been previously suggested (WO 98/54342) to use a plant IRESelement in a recombinant DNA molecule that has merely the function ofgene expression (after integration into the host genome). However, theexpression level is low. The exact reasons for this low expression levelare not known. In any event, expression is limited to the very plantcells transformed, thus the overall efficiency in whole plants isextremely low.

It has been surprisingly found that it is possible to construct a plantvector which, when introduced into a plant cell, has not only thecapability of gene expression but which has several additional functionswhich are all required for amplification and spreading throughout theplant so that the overall efficiency is extremely high. These functionscomprise infection, amplification, cell-to-cell movement andlong-distance movement. It is surprising that the required high degreeof integration of functional and structural elements on a vector doesnot impair gene expression from said vector.

The IRES element of said vector can be located upstream of saidnon-viral gene to be expressed for directly supporting its translation.Alternatively, said IRES element may indirectly support the translationof said gene to be expressed by directly supporting the translation ofanother gene essential for a function of said vector selected from thegroup of infection, amplification and cell-to-cell or long-distancemovement of said vector.

It is a further object to provide a vector which is capable of theeffective suppression of a gene in a plant. This object has beenachieved by a vector capable of amplification in a plant comprising anucleic acid having or coding for at least one IRES element necessaryfor translation of a gene required for amplification of said vector andlocated downstream of said IRES element, said vector further comprisingat least a portion of a sequence of the host plant genome in ananti-sense orientation for suppressing a gene of the host plant.

Further preferred embodiments are defined in the subclaims.

Here, the first plant expression and amplification vectors based onplant active translational (IRES) elements are described. Existing IRESelements isolated from animal viruses do not support translation inplant cells. Therefore, knowledge accumulated in animal expressionsystems is not applicable to plants. Animal IRES elements have neverbeen tested for other functional properties, such as residual promoteractivity, so this invention discloses the first bona fide cases of geneexpression in plants relying exclusively on translation rather than ontranscription with a subgenomic promoter necessary for expression of agene downstream thereof.

The vectors of this invention allows preferably for regulation andpreferential expression of a gene of interest in a plant by suppressingcap-dependent translation. In another preferred embodiment, very shorthomologous or artificial IRES elements are used, thus adding to thestability of the resulting vectors.

A preferred advantage of this strategy is that IRES sequences can beinserted upstream or downstream of viral gene(s) (e.g. the coat proteingene of tobacco mosaic virus such that translation of downstream foreigngene(s) or the viral gene(s), respectively, may occur viacap-independent internal ribosome entry pathway. Thus, saidcap-independent translation of foreign gene(s) will occur frombicistronic or/and polycistronic RNAs.

General Problem Situation and Definitions

Upon infection of a plant with a virus the early events of viralinfection (entry and genome uncoating) occur. Then the virus must engagein activities that enable its genome to be expressed and replicated. Theviral genome may consist of one (monopartite) or more (multipartite) RNAor DNA segments, and each of these segments may under certain conditionsbe capable of replicating in the infected cell. A viral replicon hasbeen defined as “a polynucleotide of viral sequences that is replicatedin host cells during the virus multiplication cycle” (Huisman et al.,1992, “Genetic engineering with plant viruses”, T. M. A. Wilson and J.W. Davies eds., 1992, CRC Press, Inc.). In this invention we use theterm “amplification-based expression system” to designate either afull-length viral genome or any fragment of viral RNA or DNA that (i)contains and is able to express foreign sequences, non-native for thewild-type parental virus (ii) replicates either by itself or as a resultof complementation by a helper virus or by a product of the transgenicplant host. The terms “amplification-based expression system” and“recombinant viral vector” are closely similar. These systems representa recombinant nucleic acid containing additional sequences, homologous(native) or foreign, heterologous (non-native) with respect to the viralgenome. The term “non-native” means that this nucleic acid sequence doesnot occur naturally in the wild-type genome of the virus and originatesfrom another virus or represents an artificial synthetic nucleotidesequence. Such an amplification-based system derived from viral elementsis capable of replicating and, in many cases, cell-to-cell as well aslong-distance movement either in a normal or/and in a geneticallymodified transgenic host plant. In the latter case the transgenic plantshould complement the viral components of a vector which may bedeficient in a certain function, i.e. the product(s) of a transgeneessential for vector replication and/or expression of its genes orlong-distance transport should be provided by the transgenic plant.

Plant virus amplification-based vectors based on the monopartite (e.g.tobacco mosaic virus, TMV) or multipartite (e.g. members of Bromoviridaefamily) genome have been shown to express foreign genes in host plants(for review, see “Genetic engineering with plant viruses”, T. M. A.Wilson and J. W. Davies eds.,1992, CRC Press, Inc.).

The majority (about 80%) of known plant viruses contains plus-sensesingle-stranded RNA (ssRNA) genomes that are infectious when beingisolated from the virions in a form of free RNA. This means that at thefirst step of the virus replication cycle, genomic RNA must betranslated in order to produce the virus-specific RNA-dependent RNApolymerase (replicase) that is absent from uninfected plant cells and,therefore, is essential for viral RNA replication (for review, see Y.Okada 1999, Philosoph. Transact. of Royal Soc., B, 354, 569-582). Itshould be mentioned that plus-sense ssRNA viruses differ in translationstrategies used for genome expression: the genomes of so calledpicoma-like viruses represent a single continuous open reading frame(ORF) translated by the ribosome into a large polyprotein which is thenproteolytically processed into functionally active virus-coded proteins.The virus-specific proteinase(s) are involved in polyprotein processing.A second peculiar feature of picorna-like viruses is that their genomicRNA contains, instead of cap structure, a small viral protein covalentlylinked to the 5′-end of the genome.

In this invention we most preferably focus on viruses of the so-calledSindbis-like superfamily that comprises many plant viruses, inparticular, more than a dozen of viruses belonging to the genusTobamovirus (for review, see A. Gibbs, 1999, Philosoph. Transact. ofRoyal Soc., B, 354, 593-602). The technology ensures cap-independent andviral promoter-independent expression of foreign genes.

The genome of tobamoviruses (TMV U1 is the type member) contains fourlarge ORFs. The two components of the replicase (the 130-kDa and itsreadthrough 183-kDa proteins) are encoded by the 5′-proximal region ofthe genomic RNA and are translated directly from genomic RNA. The3′-terminal 15 nucleotides of the 180-kDa protein gene of TMV U1 overlapwith the ORF coding for the 30-kDa protein responsible for cell-to-cellmovement of TMV infection (movement protein, MP). In TMV U1 this geneterminates two nucleotides before the initiation codon of the last genewhich encodes the 17-kDa coat protein (CP) located upstream of the3-proximal nontranslated region (3′-NTR) consisting of 204 nucleotides(in TMV U1). Translation of RNA of tobamoviruses occurs by a ribosomescanning mechanism common for the majority of eukaryotic mRNAs (forreviews, see Kozak, 1989, J. Mol. Biol. 108, 229-241; Pain, 1996 ;Merrick and Hershey,1996, In “Translational control”, eds. Hershey,Matthews and Sonenberg, Cold Spring Harbour Press, pp. 31-69; Sachs andVarani, 2000, Nature Structural Biology 7, 356-360). In accordance withthis mechanism, structurally polycistronic tobamovirus RNA isfunctionally monocistronic, i.e., only the 5′-proximal ORF encoding thereplicative proteins (130-kDa protein and its readthrough product) canbe translated from full-length genomic RNA (reviewed by Palukaitis andZaitlin,1986, In “The Plant Viruses”, van Regermortel andFraenkel-Conrat eds., vol.2, pp.105-131, Plenum Press, NY). It should beemphasized that the 68-nucleotide 5′-terminal nontranslated leadersequence of TMV U1 termed omega (Ω) has been shown to play the role ofan efficient translational enhancer stimulating the translation of the5′-proximal ORF.

The 5-distal MP and CP genes are translationally silent in full-lengthTMV U1 RNA, however, they are translated from separate mRNAs referred toas subgenomic RNAs (sgRNA). Apparently, the tobamovirus sgRNAs aretranscribed from negative-sense genomic RNA and share a common3′-terminus. The expression of TMV genes that are translated from sgRNAsis regulated independently, both quantitatively and temporarily: the MPis produced transiently during early steps of infection and accumulatesto relatively low levels (about 1% of total plant protein), whereas theCP constitutes up to 70% of total plant protein synthesis and the CP canaccumulate up to 10% of total cellular protein (Fraser, 1987, In“Biochemistry of virus-infected plants”, pp.1-7, Research Studies PressLtd., Letchworth, England).

It is clear that production of each sgRNA is controlled by differentcis-acting sequences termed “subgenomic mRNA promoter” (sgPR).Generally, this term indicates the region of the viral genome(presumably in a minus-sense RNA copy) that can be recognized by thereplicase complex to initiate transcription from the internally locatedsgPR sequence to produce sgRNA. However, for convenience, by the term“subgenomic promoter” we conventionally mean a nucleotide sequence inplus-sense viral RNA that is usually located upstream of the codingsequence and the start point of sgRNA and which is functionally involvedin the initiation of the sgRNA synthesis. However, it should be takeninto consideration that some viral sgPRs are located not only upstreamof the controlled viral gene, but can even overlap with this gene(Balmori et al., 1993, Biochimie (Paris) 75, 517-521). Each sgPRoccupies a different position in the TMV genome. None of the sgPRs ofTMV has been precisely mapped, but the 250 nucleotides upstream of theCP gene have been shown to promote synthesis of the CP sgRNA (Dawson etal., 1989, Virology 172, 285-292). Lehto et al. (1990, Virology 174,145-157) inserted in the TMV genome (in front of the MP gene) sequences(253 and 49 nucleotides) preceding the CP gene in order to estimate thesize of the CP sgPR. The insertion did not remove the native MP sgPR,but separated it from the MP ORF. The mutant (called KK6) with aninserted 253 nt promoter region replicated stably and moved systemicallyover the infected plant. It is not unexpected that in the KK6 mutant theinsertion changed the length of the MP sgRNA leader (Lehto et al., 1990,Virology 174, 145-157) (see FIG. 18). The KK6 MP sgRNA leader was 24nucleotides compared to 9 b.p. for the CP sgRNA.

By contrast, the mutant with an inserted 49-nt fragment of the promoterregion replicated only transiently before being overtaken by a progenyof wild-type virus with the insert deleted. In addition, it has beenshown (Meshi et al., 1987, EMBO J., 6, 2557-2563) that production of theCP sgRNA was reduced when the 96-nt region derived from CP sgPR wasused. It is concluded that the 49-96 nt sequences upstream of the CPgene did not contain the entire sgPR of the TMV U1 CP gene, whereas the250-nt sequence included complete sgPR.There is little information aboutthe structure and mapping of sgPR controlling the expression of the TMVMP gene. Because the putative MP sgPR sequence overlaps with the 183-kDareplicase protein, the mutational analysis of the MP sgPR wascomplicated. Preliminary results of W. Dawson and co-workers reportedrecently delineated the boundaries of the minimal and full MP sgPR ofTMV U1 (Grdzelishvili et al., 2000, Virology 276, in press). Computerfolding of the region upstream of the MP gene reveals two stem-loopstructures, located 5′-proximally to the 75-nt region preceding AUGcodon of the MP gene.

It is assumed that in contrast to genomic RNA and the CP sgRNA, thesgRNA of the MP gene (so called I₂ sgRNA) is uncapped (for review see:Okada, 1999, Philosoph. Transact. Of Royal Soc., B, 354, 569-582). Thepresent invention provides the results confirming the absence of thecap-structure in I₂ sgRNAs of both TMV U1 and crTMV (FIG. 16).

It has been shown by W. Dawson with colleagues that an important factoraffecting the expression of a foreign gene from the vector virus is theposition of the foreign gene relative to the 3′-terminus of viralgenome: the efficiency of expression increased dramatically when thegene was placed closer to the 3′-terminus (Culver et al., 1993, Proc.Natl. Acad. Sci. USA 90, 2055-2059). The highest expressed gene is thatof the CP which is adjacent to the 3′-NTR that consists (in TMV U1 RNA)of three pseudoknots followed by a tRNA-like structure. It was suggested(Shivprasad et al., 1999, Virology 355, 312-323) that the proximity ofthe gene to the pseudoknots rather than to the 3-terminus was the mainfactor increasing expression of the foreign gene. Many important aspectsof the TMV sg PRs structure were clarified due to the efforts of W.Dawson's group, however, the general conclusion of these authors wasthat “we are still in the empirical stage of vector building”(Shivprasad et al., 1999, Virology 355, 312-323).

The above shows that the synthesis of sgRNAs is essential for expressionof the 5′-distal genes of TMV genome, since these genes aretranslationally silent in full-length RNA. The mechanism of geneautonomization by subgenomization can be regarded as a strategy used byTMV in order to overcome the inability of eukaryotic ribosomes toinitiate translation of the 5′-distal genes from polycistronic mRNA.According to the traditional ribosome scanning model (Kozak, 1999, Gene234, 187-208), the internal genes of a polycistronic eukaryotic mRNA arenot accessible to ribosomes.

Recently, we have isolated a crucifer infecting tobamovirus (crTMV) fromOleracia officinalis L. plants. A peculiar feature of crTMV was itsability to infect systemically members of Brassicaceae family. Inaddition, this virus was able to systemically infect plants of theSolanaceae family and other plants susceptible to TMV U1. The genome ofcrTMV (6312 nucleotides) was sequenced (Dorokhov et al., 1994, FEBSLetters 350, 5-8) and was shown to contain four traditional ORFsencoding proteins of 122-kDa (ORF1), 178-kDa (ORF2), the readthroughproduct of 122-kDa protein, a 30-kDa MP (ORF3), and a 17-kDa CP (ORF4).A unique structural feature of crTMV RNA was that, unlike othertobamoviruses, the coding regions of the MP and CP genes of crTMV areoverlapped by 75 nucleotides, i.e. the 5′-proximal part of the CP codingregion also encodes the C-terminal part of the MP.

In order to provide a clear and consistent understanding of thespecification and the claims, including the scope given herein to suchterms, the following definitions are provided:

Adjacent: A position in a nucleotide sequence immediately 5′ or 3′ to adefined sequence.

Amplification vector: A type of gene vector that, upon introduction intoa host cell, is capable of replicating therein.

Anti-Sense Mechanism: A type of gene regulation based on controlling therate of translation of mRNA to protein due to the presence in a cell ofan RNA molecule complementary to at least a portion of the mRNA beingtranslated.

Chimeric Sequence or Gene: A nucleotide sequence derived from at leasttwo heterologous parts. The sequence may comprise DNA or RNA.

Coding Sequence: A deoxyribonucleotide sequence which, when transcribedand translated, results in the formation of a cellular polypeptide or aribonucleotide sequence which, when translated, results in the formationof a cellular polypeptide.

Compatible: The capability of operating with other components of asystem. A vector or plant viral nucleic acid which is compatible with ahost is one which is capable of replicating in that host. A coat proteinwhich is compatible with a viral nucleotide sequence is one capable ofencapsidating that viral sequence.

Gene: A discrete nucleic acid sequence responsible for a discretecellular product.

Gene to be expressed: A gene of technological interest to be expressed.

Host: A cell, tissue or organism capable of replicating a vector orplant viral nucleic acid and which is capable of being infected by avirus containing the viral vector or plant viral nucleic acid. This termis intended to include procaryotic and eukaryotic cells, organs, tissuesor organisms, where appropriate.

Host Plant Genome: This term mean preferably the nuclear genome of ahost plant cell, but may also include mitochondrial or chloroplast DNA.

Infection: The ability of a virus or amplification-based vector totransfer its nucleic acid to a host or introduce nucleic acid into ahost, wherein the viral nucleic acid or a vector is replicated, viralproteins are synthesized, and new viral particles assembled. In thiscontext, the terms “transmissible” and “infective” are usedinterchangeably herein.

Internal Ribosome Entry Site (IRES) element, or IRES: a nucleotidesequence of viral, cellular or synthetic origin, which at the stage oftranslation is responsible for internal initiation.

IRES element necessary for translation of a gene downstream thereof:IRES element which is effective for translation of said gene in thesense that without such IRES element no technologically significanttranslation of this gene will occur.

Non-viral gene: A gene not functional for the life cycle of a virus.

Phenotypic Trait: An observable property resulting from the expressionof a gene.

Plant Cell: The structural and physiological unit of plants, consistingof a protoplast and the cell wall.

Plant Organ: A distinct and visibly differentiated part of a plant, suchas root, stem, leaf or embryo.

Plant Tissue: Any tissue of a plant in planta or in culture. This termis intended to include a whole plant, plant cell, plant organ,protoplast, cell culture, or any group of plant cells organized into astructural and functional unit.

Production Cell: A cell of a tissue or organism capable of replicating avector or a viral vector, but which is not necessarily a host to thevirus. This term is intended to include prokaryotic and eukaryoticcells, organs, tissues or organisms, such as bacteria, yeast, fungus andplant tissue.

Promoter: The 5′-non-coding sequence upstream to and operationallyconnected to a coding sequence which is involved in the initiation oftranscription of the coding sequence.

Protoplast: An isolated plant cell without cell walls, having thepotency of regeneration into cell culture or a whole plant.

Recombinant Plant Viral Nucleic Acid: Plant viral nucleic acid which hasbeen modified to contain nonnative nucleic acid sequences.

Recombinant Plant Virus: A plant virus containing the recombinant plantviral nucleic acid.

Reporter Gene: A gene the gene product of which can be easily detected.

Subgenomic Promoter (sgPR): A promoter of a subgenomic mRNA of a vectoror a viral nucleic acid.

Substantial Sequence Homology: Denotes nucleotide sequences that arehomologous so as to be substantially functionally equivalent to oneanother. Nucleotide differences between such sequences havingsubstantial sequence homology will be de minimus in affecting functionof the gene products or an RNA coded for by such sequence.

Transcription: Production of an RNA molecule by RNA polymerase as acomplementary copy of a DNA sequence.

Translation: Production of a polypeptide by a ribosome (frequently bymeans of scanning a messenger RNA).

Vector: A nucleic acid, which is capable of genetically modifying a hostcell. The vector may be single-stranded (ss) (+), ss (−) ordouble-stranded (ds).

Virus: An infectious agent composed of a nucleic acid encapsidated in aprotein. A virus may be a mono-, di-, tri- or multi-partite virus.

Advantages of the Invention

This invention provides a novel strategy for constructing theamplification-based vectors for foreign (heterologous, non-native) geneexpression such that translation of these genes can occur through anIRES-mediated internal ribosome entry mechanism from a polycistronic RNAand/or through IRES-mediated cap-independent internal ribosome entrymechanism from bi- and multicistronic sgRNA produced from the vector inthe infected cell. In either event, the IRES element is necessary fortranslation of a gene. One of the advantages of this strategy is that itdoes not require any specific manipulation in terms of sgPRs: the onlysequences that should be inserted into the vector are theIRES-sequence(s) (native or/and non-native) upstream of gene(s) to betranslated. As a result, translation of downstream gene(s) is promotedby the inserted IRES sequences, i.e. is cap-independent. The sequencesegment harboring an IRES element preferably does not function assubgenomic promoter to a technically significant degree. This means thatthis sequence segment either does not cause any detectable production ofcorresponding subgenomic RNA or that for the translation of any suchsubgenomic RNA, if formed by any residual subgenomic promoter activityof said sequence segment, this IRES element is still necessary for thetranslation of a downstream gene. Consequently, in a special case,primary recombinant RNA produced by the vector comprises: one or morestructural genes preferably of viral origin, said IRES sequence, the(foreign) gene of interest located downstream of the IRES and the3′-NTR. It is important that this strategy allows a simultaneousexpression of more than one foreign gene by insertion of a tandem of two(or more) foreign genes, each being controlled by a separate IRESsequence. The present invention is preferably directed to nucleic acidsand recombinant viruses which are characterised by cap- independentexpression of the viral genome or of its subgenomic RNAs or ofnon-native (foreign) nucleic acid sequences and which are capable ofexpressing systemically in a host plant such foreign sequences viaadditional plant-specific IRES element(s).

In a first preferred embodiment, a plant viral nucleic acid is providedin which the native coat protein coding sequence and native CPsubgenomic promoter have been deleted from a viral nucleic acid, and anon-native plant viral coat protein coding sequence with upstreamlocated plant virus IRES element has been inserted that allows forcap-independent expression in a host plant, whereas packaging of therecombinant plant viral nucleic acid and subsequent systemic infectionof the host by the recombinant plant viral nucleic acid are maintained.

The recombinant plant viral nucleic acid may contain one or moreadditional native or non-native IRES elements that function astranslation elements and which have no transcriptional activity, i.e.are effecticely unable to function as a subgenomic promoter. Each nativeor non-native IRES element is capable of providing cap-independentexpression of adjacent genes or nucleic acid sequences in the hostplant.

In a second preferred embodiment, an amplification and expression vectoris provided in which native or non-native plant virus IRES element(s)located upstream of foreign nucleic acid sequences are inserteddownstream of a native coat protein gene. The inserted plant virus IRESelement may direct cap-independent expression of adjacent genes in ahost plant. Non-native nucleic acid sequences may be inserted adjacentto the IRES element such that said sequences are expressed in the hostplant under translational control of the IRES element to synthesize thedesired product.

In a third preferred embodiment, a recombinant vector nucleic acid isprovided as in the second embodiment except that the native ornon-native plant viral IRES element(s) with downstream located foreignnucleic acid sequences are inserted upstream of native coat proteinsubgenomic promoter and coat protein gene.

In a fourth preferred embodiment, a recombinant vector nucleic acid isprovided in which native or non-native plant viral IRES element(s) is(are) used at the 5′ end of the viral genome or in the viral subgenomicRNAs so as to render translation of a downstream gene(s)cap-independent.

In a fifth preferred embodiment, inhibition of cap-dependent translationis being utilised to increase the level of cap-independent translationfrom said vectors.

The viral-based amplification vectors are encapsidated by the coatproteins encoded by the recombinant plant viral nucleic acid to producea recombinant plant virus. The recombinant plant viral nucleic acid iscapable of replication in the host, systemic spreading in the host, andcap-independent expression of foreign gene(s) or cap-independentexpression of the whole viral genome or of subgenomic RNAs in the hostto produce the desired product. Such products include therapeutic andother useful polypeptides or proteins such as, but not limited to,enzymes, complex biomolecules, or polypeptides or traits or productsresulting from anti-sense RNA production. Examples for desirable inputtraits are resistance to herbicides, resistance to insects, resistanceto fungi, resistance to viruses, resistance to bacteria, resistance toabiotic stresses, and improved energy and material utilization. Examplesfor desirable output traits are modified carbohydrates, modifiedpolysaccharides, modified lipids, modified amino acid content andamount, modified secondary metabolites, and pharmaceutical proteins,including enzymes, antibodies, antigens and the like. Examples for traitregulation components are gene switches, control of gene expression,control of hybrid seed production, and control of apomixis.

The present invention is also directed to methods for creation ofartificial, non-natural IRES elements (as opposed to IRESs isolated fromliving organisms) providing cap-independent and promoter independentexpression of a gene of interest in plant cells (and perhapsadditionally in yeast or animal cells). Examples for living organismsfrom which IRESs may be isolated are animal viruses and plant viruses.Examples for animal viruses are hepatitis C virus, infectious bronchitisvirus, picornaviruses such as poliovirus and encephalomiocarditis virus,and retroviruses such as moloney murine leukemia virus, and harveymurine sarcoma virus. Examples for plant viruses are potato virus X,potyviruses such as potato virus Y and turnip mosaic virus,tobamoviruses such as crucifer-infecting tobamovirus, and comovirusessuch as cowpea mosaic virus. Alternatively, natural IRESs may beisolated from cellular messenger RNAs like those derived fromantennapedia homeotic gene, human fibroblast growth factor 2, andtranslation initiation factor elF-4G.

In a sixth preferred embodiment, artificial, non-natural IRES elementsare created on the basis of complementarity to 18S rRNA of eukaryoticcells, including yeast, animal and plant cells. In a seventh preferredembodiment, artificial, non-natural IRES elements are created on thebasis of repeated short stretches of adenosin/guanosin bases.

In an eighth preferred embodiment of this invention, a method ofengineering and using viral-based amplification vectors is presented,wherein viral genome expression in plant cells occurs under the controlof a plant-specific artificial transcription promoter.

In a ninth preferred embodiment of the present invention, a method ofconstruction and using viral-based amplification vectors is presented,which vectors allow for expression from replicons being formed in plantcells as a result of primary nuclear transcript processing.

In a tenth preferred embodiment of this invention, a procedure isdescribed for using circular single-stranded viral-based amplificationvectors for cap-independent expression of foreign genes in plants.

In an eleventh preferred embodiment of the present invention, methodsare presented that allow for expression of a gene of interest in cellsunder conditions favoring cap-independent translation. In one example,cells infected with an amplification vector are treated with a compoundinhibiting cap-dependent translation. In another example, the vectoritself contains a gene, the product of which has an inhibiting effect oncap-dependent translation in the host or an anti-sense sequence havingsaid function.

In a twelvth preferred embodiment of this invention, a method isdescribed that allows, by using in vivo genetic selection, to identifyan IRES sequence that provides cap-independent expression of gene ofinterest or a reporter gene in an expression vector.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 10 depicts vectors T7/crTMV and SP6/crTMV.

FIG. 11 depicts vectors T7/crTMV/IRES_(MP,75) ^(CR)-GUS,T7/crTMV/IRES_(MP,75) ^(UI)-GUS, T7/crTMV/IRES_(MP,228) ^(CR)-GUS,T7/crTMV/IRES_(CP,148) ^(CR)-GUS, T7/crTMV/SPACER_(CP,148) ^(UI)-GUS andT7/crTMV/PL-GUS.

FIG. 12. Mapping of the 5′end of the crTMV I₂ sgRNA by primer extention(A) and putative secondary structure of 12 sgRNA 5′NTR.

FIG. 13. crTMV 12 sgRNA 5′NTR contains translation inhibiting hairpinstructure. (A)-depicts artificial transcripts used for in vitrotranslation in wheat germ extracts (WGE); (B)-shows translation productssynthesized in WGE.

FIG. 14. Tobamoviruses contain a putative translation inhibiting hairpinstructure upstream of the MP gene.

FIG. 15. Method of the specific detection of capped mRNAs. A, B. RNA-tagwith known sequence is ligated specifically to the cap of tested RNA. C.Reverse transcription with 3′-specific primer and synthesis of firststrand of cDNA. Tag sequence is included to the sequence of cDNA. D. PCRwith tag-specific and 3′-specific primers. The appearance of therespective PCR band indicates the presence of cap-structure in thetested RNA. E. PCR with 5′-specific and 3′-specific primers. Theappearance of PCR band serves as a control for PCR reaction andindicates a presence of the specific tested RNA in the reaction. FRelative comparison of the lengths of obtained PCR bands.

FIG. 16 a and 16 b. Detection of the presence of a cap-structure at the5′-terminus of viral RNAs in a 2% agarose gel. Arrows indicate therespective PCR bands.

FIG. 17. depicts KK6-based TMV vectors.

FIG. 18. Nucleotide sequence of 5′NTR of KK6 and KK6-IRES_(MP,75) ^(CR)I₂sgRNA.

FIG. 19. Time-course of CP and MP accumulation in leaves inoculated withKK6-IRES_(MP,75) ^(CR) (K86), KK6 and TMV UI.

FIG. 20. CP accumulation in tobacco infected with KK6, KK6-IRES_(MP,75)^(CR), KK6-IRES_(MP,125) ^(CR), and KK6-H-PL and KK6-PL.

FIG. 21 depicts a crTMV IRESmp multimer structure and complementarity to18S rRNA.

FIG. 22 depicts bicistronic transcripts containing IRES_(MP,75) ^(CR)the tetramers of 18-nt segment of IRES_(CP,148) ^(CR), 19-nt segment ofIRES_(MP,75) ^(CR), polylinker (PL) as intercistronic spacer andproducts of their translation in RRL.

FIG. 23 depicts the IRES_(CP,148) ^(CR) structure.

FIG. 24 depicts constructs used for IRES_(CP,148) ^(CR) sequenceelements testing in vitro and in vivo.

FIG. 25. GUS activity testing in WGE after translation of transcriptsdepicted in FIG. 30.

FIG. 26. GUS activity test in tobacco protoplasts transfected with 35Spromoter-based constructs analogous to those depicted on FIG. 30.

FIG. 27 depicts a scheme of cloning of two infectious TMV vectorscontaining IRES_(MP,75) ^(CR) in 5′NTR.

FIG. 28 depicts vector Act2/crTMV.

FIG. 29 depicts pUC-based vector Act2/crTMV/IRES_(MP,75) ^(CR)-GUS.

FIG. 30 depicts circular single-stranded vectorKS/Act2/crTMV/IRES_(MP,75) ^(CR)-GUS.

FIG. 31 depicts vector KS/Act2/crTMV/IRES_(MP,75) ^(CR)-GUS

FIG. 32 depicts construct 35S/CP/IRES_(MP,75) ^(CR)/GUS.

FIG. 33 depicts construct 35S/GUS/IRES_(MP,75) ^(CR)/CP.

FIG. 34 depicts construct 35S/CP-VPg/IRES_(MP,75) ^(CR)/GUS.

FIG. 35 shows a construct for in vivo genetic selection to identify aviral subgenomic promoter or an IRES sequence that providescap-independent expression of a gene of interest in an expressionvector.

DETAILED DESCRIPTION OF THE INVENTION

A primary objective of this invention is to provide a novel strategy forthe construction of amplification-based vectors for foreign(heterologous, non-native) gene expression such that translation ofthese genes will occur by virtue of IRES-mediated cap-independentinternal ribosome entry mechanism from polycistronic genomic viral RNAsand/or from bi- and multicistronic sgRNAs produced by an amplificationvector, preferably a viral vector in a plant cell.

Construction of recombinant plant viral RNAs and creation ofamplification-based vectors for the introduction and expression offoreign genes in plants has been demonstrated by numerous authors usingthe genomes of viruses belonging to different taxonomic groups (forreview, see “Genetic Engineering With Plant Viruses”, 1992, eds. Wilsonand Davies, CRC Press, Inc.). Tobamoviruses are considered to beconvenient subjects for the construction of viral vectors. Donson et al.(U.S. Pat. Nos. 5,316,931; 5,589,367 and 5,866,785) created TMV-basedvectors capable of expressing different foreign genes in a host plant.Thus, neomycin phosphotransferase, a-trichosantin and several otherforeign genes were inserted adjacent to the subgenomic promoter (sgPR)of TMV CP. Donson et al., (1993, PCT WO 93/03161) developed on the basisof a tobamovirus “a recombinant plant viral nucleic acid comprising anative plant viral subgenomic promoter, at least one non-native plantviral subgenomic promoter and a plant viral coat protein codingsequence, wherein said non-native plant viral subgenomic promoter iscapable of initiating transcription of an adjacent nucleic acid sequencein a host plant and is incapable of recombination with the recombinantplant viral nucleic acid subgenomic promoters and said recombinant plantviral nucleic acid is capable of systemic infection in a host plant”.

Contrary to the technology of Donson et al., the present invention isnot concerned with sgPRs in order to construct a viral replicon-basedplant expression system. Instead of sgPRs, our technology manipulateswith IRES-sequences of different origin (native or non-native for thevirus), the sequences of which effectively lack sgPR activity, i.e. areeffectively unable to promote sgRNA production. Therefore, these IRESsequences should not be regarded as sgPRs even in the case theyrepresent a nonfunctional segment of a sgPR.

It is generally believed that uncapped transcripts of full-length viralRNA obtained after in vitro transcription of cDNA clones are generallynon-infectious for intact plants and isolated protoplasts. Therefore,capping of a virus expression vector RNA transcript is generallyconsidered as a prerequisite for in vitro transcript infectivity. CappedRNA transcripts are commonly used for introducing a viral vector RNAinto a plant. It is important to note that in some cases viral RNA maybe encapsidated by the coat protein using a simple procedure of in vitroassembly. Thus, TMV virions as well as pseudovirions containing vectorRNA can be readily produced from CP and in vitro transcripts or purifiedauthentic viral RNA. About fifteen years ago, it has been shown by Meshiet al. (1986, Proc. Natl. Acad. Sci. USA 85, 5043-5047) that (1) theuncapped transcripts of full-length TMV RNA produced in vitro areinfectious in the absence of a cap analogue, although their specificinfectivity is very low.

In the present invention, uncapped expression vector RNA reassembledwith TMV CP can be used for plant inoculations in order to overcome itslow infectivity. At least one of the additional approaches described inthis invention opens the technical possibilities for plant infectionwith a cap-independent plant viral vector. This is the method ofinsertion of a full-length single-stranded (ss) DNA copy of a viralvector under control of an appropriate DNA promoter. After inoculationof a host plant with the recombinant viral DNA, the infectiousfull-length RNA of a plant viral vector, which will be able to replicateand spread over the plant, will be produced. In other words, theseprocedures, taken together with the fact of cap-independent expressionof foreign gene(s) promoted by IRES sequences, make both processes,namely host plant inoculation and foreign gene expression, entirelycap-independent.

An important preferred object of the present invention is the creationof a series of crTMV genome-based viral vectors with the “IRES-foreigngene” block inserted between the CP gene and 3′-NTR. Various IRES andcontrol sequences were used (see FIG. 11) in combination with twodifferent reporter genes (GUS and GFP). A unique feature of thisinvention is that the foreign genes that were located outside of theviral sgPR sequences were expressed in the infected plantcap-independently from the 3′-proximal position of genomic and sgRNAsproduced by the vector. In particular, the IRES_(MP,75) ^(CR) sequencerepresenting the 3′-terminal part of the 5′-nontranslated leadersequence of crTMV sgRNA I₂ was efficient in mediating cap-independentexpression of the 3′-proximal foreign gene in plants infected with aviral vector. It should be emphasized that said crTMV-based viralvectors produce three types of viral plus-sense ssRNAs in infectedplants, including: i) full-length genomic RNA, ii) tricistronic I₂ sgRNA(our data show that the latter sgRNA is uncapped, contrary tofull-length RNA), and iii) bicistronic sgRNA containing the first CPgene and the second foreign gene. Therefore, all these RNAs are3′-coterminal and cap-independent translation of their 3′-proximal genefrom either capped (full-length and bicistronic) or uncapped(tricistronic) RNAs is promoted by the preceding IRES sequence.

An important characteristic of virus-based vectors is their stability.However, the TMV-based vectors with foreign genes usually do not moveefficiently through phloem in plants that can be systemically infectedwith wild-type virus. This may be due to increased length of therecombinant viral RNA and/or to the presence of the repeated sequences,which could lead to recombinations and deletions resulting in reversionsto wild-type virus. The conversion of the progeny population towild-type virus occurs in systemically infected leaves.

An important characteristic for a virus-based vector is the level offoreign protein gene expression and the level of protein accumulation.The vector is able to produce readily visible bands corresponding to GUSstained in SDS-PAGE.

The technologies suitable for construction of amplification-basedvectors capable of expressing foreign sequences in host plants have beendeveloped on the basis of different viral genomes (e.g., see G.Della-Cioppa et al., 1999, PCT WO 99/36516). The central feature ofthose inventions was that the recombinant plant viral nucleic acid“contains one or more non-native subgenomic promoters which are capableof transcribing or expressing adjacent nucleic acid sequences in thehost plant. The recombinant plant viral nucleic acids may be furthermodified to delete all or part of the native coat protein codingsequence and to contain a non-native coat protein coding sequence undercontrol of the native or one of the non-native plant viral subgenomicpromoters, or put the native coat protein coding sequence under thecontrol of a non-native plant viral subgenomic promoter”. In otherwords, the most important element(s) of that invention is/are the nativeand non-native sgPR sequences used for artificial sgRNAs production bythe viral vector. An important feature that distinguishes the inventionpresented by our group from others is that according to WO 99/36516, theforeign gene must be inevitably located directly downstream of the sgPRsequence, i.e. should be located at the 5′-proximal position of thechimeric sgRNA produced by the viral vector in the host plant. Bycontrast, our invention proposes that the foreign gene is separated froma sgPR (if present) at least by one (or more) viral gene(s) such thatsaid foreign gene is located 3′-proximally or internally within thefunctionally active chimeric sgRNA produced by the vector. Thus, foreigngene expression is promoted by the IRES sequence, native or non-nativeof the wild-type virus.

The next preferred object of this invention is the construction of anovel type of non-native IRES sequences, namely artificial, non-naturalsynthetic IRESs capable of promoting cap-independent translation of5′-distal genes from eukaryotic polycistronic mRNAs. We show thatintercistronic spacers complementary to 18S rRNA of varying length andcomposition are able to mediate cap-independent translation of the3′-proximal GUS gene in bicistronic H-GFP-IRES-GUS mRNA (FIG. 22).

The last but not least advantage provided by the present invention isthe possibility to combine repeats of two or more foreign genes eachbeing preceded by the native or non-native IRES sequence in theamplification-based vector genome. Expression of such a cassette of an“IRES-foreign gene” will allow the simultaneous production of two ormore foreign proteins by the vector.

Viruses belonging to different taxonomic groups can be used for theconstruction of virus-based vectors according to the principles of thepresent invention. This is right for both RNA- and DNA-containingviruses, examples for which are given in the following (throughout thisdocument, each type species name is preceded by the name of the order,family and genus it belongs to. Names of orders, families and genera arein italic script, if they are approved by the ICTV. Taxa names in quotes(and not in italic script) indicate that this taxon does not have anICTV international approved name. Species (vernacular) names are givenin regular script. Viruses with no formal assignment to genus or familyare indicated):

DNA Viruses:

Circular dsDNA Viruses:

Family: Caulimoviridae, Genus: Badnavirus, Type species: commelinayellow mottle virus, Genus: Caulimovirus, Type species: cauliflowermosaic virus, Genus “SbCMV-like viruses”, Type species: Soybeanchloroticmottle virus, Genus “CsVMV-like viruses”, Type species: Cassavavein mosaicvirus, Genus “RTBV-like viruses”, Type species: Rice tungrobacilliformvirus, Genus: “Petunia vein clearing-like viruses”, Typespecies: Petunia vein clearing virus;

Circular ssDNA Viruses: Family: Geminiviridae, Genus: Mastrevirus(Subgroup I Geminivirus), Type species: maize streak virus, Genus:Curtovirus (Subgroup II Geminivirus), Type species: beet curly topvirus, Genus: Begomovirus (Subgroup III Geminivirus), Type species: beangolden mosaic virus;

RNA Viruses:

ssRNA Viruses: Family: Bromoviridae, Genus: Alfamovirus, Type species:alfalfa mosaic virus, Genus: Ilarvirus, Type species: tobacco streakvirus, Genus: Bromovirus, Type species: brome mosaic virus Genus:Cucumovirus, Type species: cucumber mosaic virus; Family:Closteroviridae Genus: Closterovirus, Type species: beet vellows virus,Genus: Crinivirus, Type species: Lettuce infectious yellows virus,Family: Comoviridae, Genus: Comovirus Type species: cowpea mosaic virus,Genus: Fabavirus, Type species: broad bean wilt virus 1, Genus:Nepovirus, Type species: tobacco ringspot virus:

Family: Potyviridae, Genus: Potyvirus, Type species: potato virus Y,Genus: Rymovirus, Type species: ryegrass mosaic virus, Genus: Bymovirus,Type species: barley yellow mosaic virus;

Family: Sequiviridae, Genus: Sequivirus, Type species: parsnip vellowfleck virus, Genus: Waikavirus, Type species: rice tungro sphericalvirus: Family: Tombusviridae, Genus: Carmovirus, Type species: carnationmottle virus, Genus: Dianthovirus, Type species: carnation ringspotvirus, Genus: Machlomovirus, Type species: maize chlorotic mottle virus,Genus: Necrovirus, Type species: tobacco necrosis virus, Genus:Tombusvirus, Type species: tomato bushy stunt virus, Unassigned Generaof ssRNA viruses,

Genus: Capillovirus, Type species: apple stem grooving virus;

Genus: Carlavirus, Type species: carnation latent virus:

Genus: Enamovirus, Type species: pea enation mosaic virus.

Genus: Furovirus, Type species: soil-bome wheat mosaic virus. Genus:Hordeivirus, Type species: barley stripe mosaic virus, Genus:Idaeovirus, Type species: raspberry bushy dwarf virus;

Genus: Luteovirus, Type species: barley yellow dwarf virus:

Genus: Marafivirus, Type species: maize rayado fino virus:

Genus: Potexvirus, Type species: Dotato virus X;

Genus: Sobemovirus, Type species: Southern bean mosaic virus. Genus:Tenuivirus, Type species: rice stripe virus,

Genus: Tobamovirus, Type species: tobacco mosaic virus,

Genus: Tobravirus, Type species: tobacco rattle virus,

Genus: Trichovirus, Type species: apple chlorotic leaf spot virus,

Genus: Tymovirus, Type species: turnip yellow mosaic virus,

Genus: Umbravirus, Type species: carrot mottle virus,

Negative ssRNA Viruses: Order: Mononegavirales, Family: Rhabdoviridae,Genus: Cytorhabdovirus, Type Species: lettuce necrotic vellows virus,Genus: Nucleorhabdovirus, Type species: potato yellow dwarf virus;

Negative ssRNA Viruses: Family: Bunyaviridae, Genus: Tospovirus, Typespecies: tomato spotted wilt virus:

dsRNA Viruses: Family: Partitiviridae, Genus: Alphacryptovirus, Typespecies: white clover cryptic virus 1, Genus: Betacryotovirus, Typespecies: white clover cryptic virus 2, Family: Reoviridae, Genus:Fijivirus, Type species: Fiji disease virus, Genus: Phytoreovirus, Typespecies: wound tumor virus, Genus: Oryzavirus, Type species: rice raggedstunt virus;

Unassigned Viruses: Genome ssDNA: Species banana bunchy top virus,Species coconut foliar decay virus, Species subterranean clover stuntvirus,

Genome dsDNA, Species cucumber vein yellowing virus,

Genome dsRNA Species tobacco stunt virus,

Genome ssRNA Species Garlic viruses A,B,C,D, Species grapevine fleckvirus, Species maize white line mosaic virus, Species olive latent virus2, Species ourmia melon virus, Species Pelargonium zonate spot virus;

Satellites and Viroids: Satellites: ssRNA Satellite Viruses: Subgroup 2Satellite Viruses, Type species: tobacco necrosis satellite,

Satellite RNA, Subgroup 2 B Type mRNA Satellites, Subgroup 3 C Typelinear RNA Satellites, Subgroup 4 D Type circular RNA Satellites,

Viroids, Type species: potato spindle tuber viroid.

In particular, the methods of the present invention can preferably beapplied to the construction of virus replicon-based vectors using therecombinant genomes of plus-sense ssRNA viruses preferably belonging tothe genus Tobamovirus or to the families Bromoviridae or Potyviridae aswell as DNA-containing viruses. In the latter case the foreign geneshould preferably be located downstream of a viral gene and itsexpression can be mediated by the IRES sequence from bicistronic orpolycistronic mRNA transcribed by a DNA-dependent RNA polymerase from agenomic transcription promoter.

A separate preferred aspect of this invention is concerned with theapplication of the methods of the invention to the construction ofssDNA-based vectors. The geminivirus-based vectors expressing theforeign gene(s) under control of an IRES sequence can exemplify thisaspect. The geminiviruses represent a group of plant viruses withmonopartite or bipartite circular ssDNA that have twinnedquasiicosahedral particles (reviewed by Hull and Davies, 1983, Adv.Virus Res. 28, 1-45; Mullineaux et al., 1992, “Genetic engineering withplant viruses”, Wilson and Davies, eds., 1992, CRC Press, Inc.). The twossDNA components of the bipartite geminiviruses referred to as A and Bencode for 4 and 2 proteins, respectively. The DNA A contains the CPgene and three genes involved in DNA replication, whereas the DNA Bencodes for two proteins essential for the viral movement. It has beendemonstrated that the genomes of bipartite geminiviruses belonging tothe genus Begomovirus, such as tomato golden mosaic virus (TGMV) andbean golden mosaic virus (BGMV) can replicate and spread over a certainhost plant despite the deletion of the CP gene (Gardiner et al., 1988,EMBO J. 7, 899-904; Jeffrey et al., 1996, Virology 223, 208-218; Azzamet al., 1994, Virology 204, 289-296). It is noteworthy that somebegomoviruses including BGMV exhibit phloem-limitation and arerestricted to cells of the vascular system. Thus, BGMV remainsphloem-limited, while TGMV is capable of invading the mesophyll tissuein systemically infected leaves (Petty and Morra, 2000, Abstracts of19^(th) Annual meeting of American Society for Virology, p.127). Thepresent invention proposes to insert the foreign gene in a bipartitegeminivirus genome by two ways: (i) downstream of one of its (e.g.,BGMV) genes, in particularthe CP gene such that the CP ORF will beintact or 3′-truncated and the IRES sequence will be inserted upstreamof the foreign gene. Therefore, the mRNA transcription will proceed fromthe native DNA promoter resulting in production of bicistronic chimericmRNA comprising the first viral gene (or a part thereof), the IRESsequence and the 3′-proximal foreign gene expression of which ismediated by the IRES. Alternatively (ii), the full-length DNA copy ofthe the RNA genome of the viral vector can be inserted into a DNA of aCP-deficient bipartite geminivirus under control of the CP genepromoter. The RNA genome of the RNA-vector-virus will be produced as aresult of DNA A transcription in the plant cell inoculated with amixture of recombinant DNA A and unmodified DNA B. An advantage of thismethod is that the geminivirus-vector is needed as a vehicle used onlyfor delivering the vector to primary-inoculated cells: all other stepswill be performed by a tobamovirus vector itself including production ofIRES-carrying vector RNA after geminivirus-vector DNA transcription by acellular RNA polymerase, its replication, translation and systemicspread over the host plant and foreign gene(s) expression. As anadditional possibility for the creation of a ssDNA vector, cloning ofthe viral cDNA and the foreign gene into a phagemid vector andproduction of the ssDNA according to standard methods can be mentioned.

Taking into account that tobamovirus-derived IRES sequences are shown tobe functionally active in animal cells (our previous patentapplication), the methods of the present invention can be used forconstructing the recombinant viral RNAs and producing the viral vectorson the basis of animal viruses, e.g. the viruses belonging to thefamilies Togaviridae, Caliciviridae, Astroviridae, Picomaviridae,Flaviviridae in order to produce new vectors expressing the foreigngenes under control of plant virus-derived IRES sequences. Such animalvirus-based vectors for plants and animals can be useful in the fieldsof vaccine production or for gene therapy.

It should be noted, however, that the rod-like virions of Tobamovirusesand, in particular, the flexible and long virions of filamentousPotexviruses, Carlaviruses, Potyviruses and Closteroviruses apparentlyprovide the best models for realization of the methods of the presentinvention.

The next preferred objective of this invention is to use the IRESsequence in such a way that the virus-based amplification vector willcontain the IRES-sequence within its 5′-NTR. It is presumed thatinsertion of an IRES sequence does not prevent viral replication, but isable to ensure an efficient cap-independent translation of transcriptsof genomic vector RNA. Therefore, said construct may comprise: (i) AnIRES element within or downstream of the 5′-untranslated leader sequencethat is native or non-native for said viral vector and promotescap-independent translation of the viral 5′-proximal gene (the RdRp),and (ii) at least one native or non-native IRES sequence locateddownstream of one or more viral structural genes and upstream of foreigngene(s) in order to promote their cap-independent translation. Accordingto this method, the specific infectivity of uncapped full-length vectortranscripts will be increased due to efficient 5′-IRES-mediatedtranslation of the parental RNA molecules in the primary inoculatedcells.

Yet another preferred objective of the present invention is the methodof producing one or several protein(s) of interest in plant cells basedon the introduction and cap-independent expression of a foreign genefrom a mono- or polycistronic mRNA sequence mediated by the plantspecific IRES sequence located upstream of said foreign gene sequence. Aparticular feature of this method is that the technology involves aprocedure that allows to selectively switch off the cellularcap-dependent mRNA translation with the help of certain chemicalcompounds. However, this procedure does not affect the cap-independentIRES-mediated translation of mRNAs artificially introduced in the plantcells, thus allowing to control and enhance cap-independent expression.

Alternatively, the means for inhibiting the translation of cellularcapped mRNA can be applied to plants infected with said viral vectoritself that expresses the foreign gene(s) in a cap-independent manner.Under conditions when the translation of the cellular capped mRNAs isprevented, selective expression of the foreign gene(s) from said virusvector will occur. The vector of the invention may be an RNA or DNAvector. It may be ss(+), ss(−) or ds. It may show any of the modes ofamplification known from viruses. This includes the multiplication ofthe vector nucleic acid and optionally the production of coat proteinand optionally the production of proteins for cell-to-cell movement orlong-distance movement. The genes for the required replication and/orcoat and/or movement may be wholly or partially encoded in anappropriately engineered host plant. In this manner, a system isgenerated consisting of mutually adapted vector and host plant.

The vector may be derived form a virus by modification or it may besynthesized de novo. It may have only IRES elements effectively devoidof any subgenomic promoter activity. However, the vector may combine oneor several subgenomic promoters with one or several IRES elementseffectively devoid of subgenomic promoter function, so that the numberof cistrons is greater than the number of promoters.

Considering the simplest case of one IRES element, said element may belocated upstream of a (foreign) gene of interest to be expresseddirectly by said IRES element and optionally downstream of a (viral)gene for, say replication, to be expressed IRES-independent.Alternatively, the gene of interest may be upstream of an IRES elementand expressed IRES-independent and the IRES element serves for theexpression of a downstream viral gene. These simplest cases may ofcourse be incorporated singly or multiply in a more complex vector.

The vector may contain a sequence in anti-sense orientation forsuppressing a host gene. This suppression function may exist alone or incombination with the expression of a (foreign) gene of interest. Aparticularly preferred case involves the suppression of a gene essentialfor cap-dependent translation, e.g. a gene for a translation initiationfactor (e.g. elF4) associated with cap-dependent translation, so thatthe translation machinery of the host plant is wholy in service ofvector gene translation. In this case, the vector must be wholycap-independent. Of course, the vector may be generated within a plantcell from a pro-vector by the plant nucleid acid processing machinery,e.g. by intron splicing.

The IRES element may be of plant viral origin. Alternatively, it may beof any other viral origin as long as it satisfies the requirement ofoperation in a plant cell. Further, an IRES element operative in a plantcell may be a synthetic or an artificial element. Synthesis may beguided by the sequence of the 18S rRNA of the host plant, namely thesegment operative for IRES binding. It should be sufficientlycomplementary thereto. Sufficiency of complementarity can simply bemonitored by testing for IRES functionality. Complementarity in thissense comprises GC, AU and to some extent GU base pairing. Further, suchIRES element may be a multimer of such a complementary sequence toincrease efficiency. The multimer may consist of identical essentiallycomplementary sequence units or of different essentially complementarysequence units. Moreover, artificial IRES elements with high translationefficiency and effectively no subgenomic promoter activity may begenerated by a process of directed evolution (as described e.g. in U.S.Pat. No. 6,096,548 or U.S. Pat. No. 6,117,679). This may be done invitro in cell culture with a population of vectors with IRES elementsequences that have been randomized as known per se. The clones whichexpress a reporter gene operably linked to the potential IRES elementare selected by a method known per se. Those clones which showsubgenomic promoter activity are eliminated. Further rounds ofrandomization and selection may follow.

The IRES element of the vector of the invention may be effectivelydevoid of promoter activity. This means that that the expression of agene operably linked to an IRES element would not occur by a residualsubgenomic promoter activity. This mode of action may be determined bystandard molecular biology methods such as Northern blotting, primerextension analysis (Current Protocols in Molecular Biology, Ed. By F.Ausubel et al., 1999, John Wiley & Sons), 5′ RACE technology (GibcoBRL,USA), and alike. It should be added that IRES elements that showdetectable subgenomic promoter activity but operate essentially astranslational rather than transcriptional elements, are also subject ofour invention. Such discrimination could be derived, for example, bymeasuring quantitatively the relative amounts of two types of mRNAs onNorthern blots, namely the short mRNA due to sgPR activity and the longmRNA not due to sgPR activity. If the IRES element does not essentiallyoperate as a residual viral subgenomic promoter, the relative amount ofcorresponding short mRNA should be lower than 20%, preferably lower than10% and most preferably. lower than 5% of the sum of the short and longmRNA. Thus we provide as a preferred embodiment a vector capable ofamplification of a gene in a plant comprising a nucleic acid having asequence for at least one non-viral gene to be expressed and having orcoding for at least one IRES element necessary for translation of saidgene in said plant with the proviso that the expression of said gene isessentially derived from translational rather than transcriptionalproperties of said IRES element sequence when measured by standardprocedures of molecular biology.

The novel vectors of the invention open new avenues for geneticmodification of plants. As a first possibility we suggest the use fordetermining the function of a structural gene of a plant. This isnotably of interest for genomics. Therefore, a plant for which thegenome has been sequenced is of particular interest. This is a smallscale (plant-by plant) application. The vector of this invention ishighly effective for this application, since it allows suppression ofgenes of interest and/or overexpression of genes to bring out the genefunction to be discovered in an intensified manner.

In a large scale application the vector may be used to generate a traitor to produce a protein in a host plant. Infection of plants with thevector may be done on a farm field previously planted with unmodifiedplants. This allows for the first time a genetic modification of plantson a field, whereby the farmer has greatest freedom in terms ofselection of seeds and vectors from a variety of sources for producing adesired protein or trait.

Examples for plant species of interest for the application of thisinvention are monocotyledonous plants like wheat, maize, rice, barley,oats, millet and the like or dicotyledonous plants like rape seed,canola, sugar beet, soybean, peas, alfalfa, cotton, sunflower, potato,tomato, tobacco and the like.

In the following, the invention will be further described using specificexamples. Standard molecular biological techniques were carried outaccording to Sambrook et al. (1989, Molecular Cloning: a LaboratoryManual. 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y.). All plasmids utilized in the invention can be prepared accordingto the directions of the specification by a person of ordinary skill inthe art without undue experimentation employing materials readilyavailable in the art.

EXAMPLE 1

Construction of a tobamovirus Vector Infecting cruciferous Plants

Virions of a known tobamovirus called crucifer tobamovirus (crTMV) whichis able to infect systemically crucifer plants were isolated fromOlearacia officinalis L. with mosaic symptoms. Results of crTMVhost-range examination are presented in Table 1.

Plasmid Constructions

CrTMV cDNA was characterized by dideoxynucleotide sequencing (Dorokhovet al., 1994 FEBS Letters 350, 5-8). Full length T7 RNA polymerasepromoter-based infectious crTMV cDNA clones were obtained by RT-PCR fromcrTMV RNA using oligonucleotides crTMV1-Kpn5′-gcatggtaccccttaatacgactcactataGTTTAGTTTTATTGCAACAACAACAA (upstream),wherein the italic bold letters are a sequence of a Kpn I site, theunderlined lowercase letters are nucleotide sequence of the T7 RNApolymerase promoter, the uppercase letters are from the 5′-termini ofcrTMV cDNA; and crTMV2 5′-gcatgcggccgcTGGGCCCCTACCCGGGGTTAGGG(downstream), wherein the italic bold letters are sequence of NotI site,the uppercase letters are from 3′-termini of crTMV cDNA and cloning intopUC19 between KpnI and Bam HI restriction sites (FIG. 10).

Full length SP6 RNA polymerase promoter-based infectious crTMV cDNAclones were obtained by RT-PCR from crTMV RNA by using oligonucleotidescrTMV1-SP65′-gcatggtaccatttaggtgacactatagaactcGTTTTAGTTTTATTGCAACAACAACAA(upstream), wherein the italic bold letters are a sequence of a Kpn Isite, the underlined lowercase letters are a nucleotide sequence of theT7 RNA polymerase promoter, the uppercase letters are from the5′-termini of crTMV cDNA; and crTMV25′-gcatgcggccgcTGGGCCCCTACCCGGGGTTAGGG (downstream), wherein the italicbold letters are a sequence of a Not I site, the uppercase letters arefrom 3′-termini of crTMV cDNA and cloning into pUC19 between KpnI andBam HI restriction sites (FIG. 10). The full-length crTMV cDNA cloneswere characterized by dideoxynucleotide sequencing. The ability of crTMVinfectious transcripts to infect systemically Nicotiana and cruciferspecies was confirmed by infection tests on respectively Nicotianatabacum var. Samsun and Arabidopsis thaliana. TABLE 1 Virus detectionand symptoms caused by crTMV in mechanically infected plants.Non-inoculated Inoculated Leaves Upper Leaves Species Symptoms* Virus**Symptoms Virus Nicotiana tabacum L. cv. Samsun C + M + cv. Samsun NN.L + s − Nicotiana clevelandii L. L + N + M + Nicotiana glutinosa L. L +N + s − Nicotiana sylvestris L. L + N + s + Nicotiana benthamiana L. L +N + M + Nicotiana rustica L. C + M + Lycopersicum esculentum L. L + N +s − Solanum tuberosum L. s − s − Capsicum frutescens L. L + N + M +Brassica chinensis L. C + M + Brassica rapa L. C + M + Brassica napus LC + M + Brassica oleracea L. L + s − Brassica compestris L. C + M +Brassica cauliflora L. C + s − Arabidopsis thaliana L. L + N + M +Chenopodium L + N + s + amaranticolor L. Coste and Reyn. Chenopodiumquinoa L + N + s − L. Willd. Chenopodium murale L. L + N + s − Daturastramonium L. L + N + s − Plantago major L. L + N + M + Tetragoniaexpansa L. L + N + s − Beta vulgaris L. L + N + s − Petunia hybrida L.C + M + Cucumis sativus L. L + N + s − Phaseolus vulgaris L. s − s −Raphanus sativus L. s − s − Sinapis alba L. C + M 0*C, chlorosis; L, local lesion; M, mosaic; N, necrosis; s, symptomless.**Virus detected (+) or not (−) by ELISA.

EXAMPLE 2

Construction of tobamoviral Vectors for Expression of GUS Genes inNicotiana and crucifer Plants via Viral IRESs

Series of IRES-mediated expression vectors T7/crTMV/GUS were constructedas follows. First, Hind III and Xba I sites were inserted in the end ofthe CP gene of Sac II/Not I fragment of T7/crTMV vector (FIG. 10) by apolymerase chain reaction (PCR) and two pairs of specific primers.Second, IRES_(MP,75) ^(C)R-GUS, IRES_(MP,76) ^(UI)-GUS, IRES_(MP,228)^(CR)-GUS, IRES_(CP,148) ^(CR)-GUS, IRES_(CP,148) ^(UI)-GUS, PL-GUS cDNAdescribed in Skulachev et al. (1999, Virology 263, 139-154) wereinserted into Hind III and Xba I containing Sac II/Not I fragment ofT7/crTMV vector to obtain Sac I-IRES_(MP,75) ^(CR)-GUS-Not I, SacII-IRES_(MP,75) ^(UI)-GUS-Not I, Sac II-I-RES_(MP,228) ^(CR)-GUS-Not I,Sac I-IRES_(CP,148) ^(CR)-GUS-Not I, Sac II-IRES_(CP,148) ^(UI)-GUS-NotI, Sac II-PL-GUS-Not I cDNA, respectively. Third, Sac II-Not I cDNAfragment of T7/crTMV vector was replaced by Sac I-IRES_(MP,75)^(CR)-GUS-Not I or Sac II-IRES_(MP,75) ^(UI)-GUS-Not I or SacII-IRES_(MP,228) ^(CR)-GUS-Not I or Sac II-IRES_(CP,148) ^(CR)-GUS-Not Ior Sac I-IRES_(CP,148) ^(UI)-GUS-Not I or Sac II-PL-GUS-Not I cDNA toobtain respectively, vector T7/crTMV/IRES_(MP,75) ^(CR)-GUS (FIG. 11),vector T7crTMV/IRES_(MP,75) ^(UI)-GUS (FIG. 11), vectorT7/crTMV/IRESM_(MP,228) ^(CR)-GUS (FIG. 11), vectorT7/crTMV/IRES_(CP,148) ^(CR)-GUS (FIG. 11), vectorT7/crTMV/IRES_(CP,148) ^(UI)-GUS (FIG. 11 and vectorT7/crTMV/PL-GUS(FIG. 11).

EXAMPLE 3

Expression of GUS Gene in Transfected Nicotiana and crucifer Plants viaViral IRESs

This example demonstrates the tobamovirus IRES-mediated expression ofthe GUS gene in Nicotiana benthamiana and Arabidopsis thaliana plantsinfected crTMV-based vectors: T7/crTMV/IRES_(MP,75) ^(CR)-GUS (FIG. 11),vector T7/crTMV/IRES_(MP,75) ^(UI)-GUS (FIG. 11), vectorT7/crTMV/IRES_(MP,228) ^(CR)-GUS (FIG. 11), vectorT7/crTMV/IRES_(CP,148) ^(CR)-GUS (FIG. 11), vectorT7/crTMV/IRES_(CP,148) ^(UI)-GUS (FIG. 11) and vectorT7/crTMV/PL-GUS(FIG. 11).

In vitro Transcription

The plasmids T7/crTMV/IRES_(MP,75) ^(CR)-GUS (FIG. 11), vectorT7/crTMV/IRES_(MP,75) ^(UI)-GUS (FIG. 11), vector T7/crTMV/IRES_(MP,228)^(CR)-GUS (FIG. 11), vector T7/crTMV/IRES_(CP,148) ^(CR)-GUS (FIG. 11),vector T7/crTMV/IRES_(CP,148) ^(UI)-GUS (FIG. 11) andvectorT7/crTMV/PL-GUS (FIG. 11) were linearized by Not I. Therecombinant plasmids were transcribed in vitro as described by Dawson etal. (1986 Proc. Natl. Acad. Sci. USA 83, 1832-1836). Agarose gelelectrophoresis of RNA transcripts confirmed that they were intact. TheRNA concentration was quantified by agarose gel electrophoresis andspectrophotometry.

GUS Detection

Inoculated leaves were collected 10-14 days after transfection withcapped full-length transcripts. IRES activity was monitored byhistochemical detection of GUS expression as described earlier(Jefferson, 1987, Plant Molecular Biology Reporter 5, 387-405). Sampleswere infiltrated using the calorimetric GUS substrate, but the method(De Block and Debrouwer, 1992, Plant J. 2, 261-266) was modified tolimit the diffusion of the intermediate products of the reaction: 0.115M phosphate buffer, pH 7.0 containing5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-Gluc) 600 μg/ml; 3 mMpotassium ferricyanide; 10 mM EDTA. After incubation overnight at 37°C., the leaves were destained in 70% ethanol and examined by lightmicroscopy.

EXAMPLE 4 IRES_(MP,75) ^(CR) does not Function as MP Subaenomic Promoterbut Provides MP Gene Expression via Cap-Independent Internal Initiationof Translation in TMV-Infected Plants

This example uses different approaches to confirm the possibility ofIRES_(MP,75) ^(CR) used in viral vectors for cap-independent expressionof a gene of interest.

CrTMV MP Subgenomic RNA has a 125-nt Long 5′-Nontranslated Region(5′NTR) and Contains a Translation Inhibiting Stem-Loop SecondaryStructure

To determine the length and nucleotide sequence of TMV UI and crTMV MPsubgenomic RNA (I₂ sgRNA) 5′NTR, the protocol of primer extensionexperiments described by Lehto et al. (1990, Virology 174, 145-157) waschanged in the following way: (i) AMV reverse transcriptase (RT); (ii)RT reaction under 45° C.; (iii) the GC-rich primer; (iv) increased dNTPconcentration; (v) dITP to avoid secondary structure. It has been shown(FIG. 12) that the 5′UTR sequence of crTMV I₂ sgRNAs consists of 125nucleotides. This result was confirmed by direct 5′UTR RT sequencing.FIG. 12B shows that crTMV 5′NTR contains a stable hairpin-loopstructure. Being placed just upstream of the MP gene of artificialtranscript, it is able to inhibit MP gene translation in vitro (FIG.13). This means that IRES_(MP,75) ^(CR) located between 5′HI₂ ^(CR) andthe MP gene can provide efficient cap-independent internal initiation oftranslation. FIG. 14 shows that homologous to 5′HI₂ ^(CR) putativetranslation inhibiting hairpin-loop structure can be revealed in the125-nt sequence upstream of the MP gene of other tobamoviruse.

CrTMV and TMV UI MP Subgenomic RNAs are not Capped

To study the structure of the 5′-terminus of the subgenomic RNA codingfor the 30K movement protein (MP) gene of crTMV, the “Jump-Start” methodoffered by Active Motif was used. Jump-Start™ is the method of chemicalligation of an RNA tag specifically to the 5′-end of capped mRNAs.During reverse transcription, the ribo-oligonucleotide tag of a knownsequence becomes incorporated into the 3′-end of a first strand cDNA.This creates a known priming site suitable for PCR.

Initially, the 5′-terminal 2′-3′-cis-glycol groups of capped RNA wereconverted to reactive di-aldehydes via sodium periodate oxidation. 1-2μl of a tested RNA (1 μg/μl) were mixed with 14 μl of pure water and 1μl of sodium acetate buffer (pH 5.5), then 4 μl of 0.1 M sodiumperiodate were added and the reaction mixture was incubated for 1 hour.

Then a 3′-aminoalkyl derivatized synthetic ribo-oligonucleotide tag waschemically ligated to the di-aldehyde ends of oxidized RNA via reductiveamination in the presence of sodium cyanoborohydride. 5 μl of sodiumhypophosphite were added and the reaction mixture was incubated for 10minutes. Then 23 μl of water, 1 μl of sodium acetate buffer (pH 4.5) and2 μl of ribo-oligonucleotide tag 5′-CTAATACGACTCACTATAGGG (28.5 pmol/μl)were added to the reaction mixture and incubated for 15 minutes. Then 10μl of sodium cyanoborohydride were added and incubated for 2 hours. Then400 μl of 2% lithium perchlorate in acetone were added, incubated for 15minutes at −20° C. and centrifugated for 5 minutes. The pellet waswashed with acetone twice, then dissolved in 20 μl of water.

To remove an abundance of the RNA tag, CTAB precipitation in thepresence of 0.3 M NaCl was used. CTAB is a strong cationic detergentthat binds to nucleic acids to form an insoluble complex. Complexformation is influenced by the salt concentration: when the saltconcentration is above 1 M, no complex formation occurs; when it isbelow 0.2 M, all nucleic acids are efficiently included in the complex;and when between 0.3 M and 0.4 M, the incorporation of smallsingle-stranded nucleic acids into the complex is very inefficient(Belyavsky et al., 1989, Nucleic Acids Res. 25, 2919-2932; Bertioli etal., 1994, BioTechniques 16, 1054-1058). 10 μl of 1.2 M NaCl (to a finalconcentration of 0.4 M) and 3 μl of 10% CTAB (to a final concentrationof 1%) were added, the reaction mixture was incubated for 15 minutes atroom temperature and then centrifugated for 5 minutes. The pellet wasresuspended in 10 μl of NaCl, 20 μl of water and 3 μl 10% CTAB wereadded and the reaction mixture was incubated for 15 minutes at roomtemperature and then centrifugated for 5 minutes. The pellet wasdissolved in 30 μl of 1.2 M NaCl, 80 μl of 96% ethanol was added, andthe reaction mixture was incubated overnight at −20° C. Then it wascentrifugated for 5 minutes and washed with 70% ethanol. Then the pelletof tagged RNA was dissolved in 24 μl of water.

Finally, reverse transcription with 3′-gene specific primers resulted inincorporation of the 5′-tag sequence at the 3′-terminus of first-strandcDNA. For reverse transcription, 12 μl of tagged RNA, 1 μl of specific3′-end primers, 4 μl of 5× buffer for SuperScript™II (Gibco BRL LifeTechnologies) containing 250 mM Tris-HCl (pH 8.3), 375 mM KCl, 15 mMMgCl₂ were mixed and heated at 95° C. for 30 seconds, then cooled onice. Then to the reaction mixture 0.5 μl of DTT (to 1 mM finalconcentration), 2 μl of 10 mM dNTP, 0.5 μl of RNAsine, 0.5 μl ofSuperScript™II were added and incubated for 1 hour at 42° C. Then 1 μlof 40 mM MnCl₂ was added and the reaction mixture was incubated for 15minutes at 42° C. The presence of MnCl₂ in the reaction mixture allowsSuperScript™ to overcome the cap structure during reverse transcriptionmore efficiently: when using 3 mM MgCl₂ and 2 mM MnCl₂, the reversetranscriptase was shown to reveal an extraordinary high cap-dependenttransferase activity, and typically the enzyme added preferentiallythree or four cytosine residues in the presence of 5′-capped mRNAtemplates (Chenchik et al., 1998, Gene cloning and analysis by RT-PCR,edited by Paul Siebert and James Larrick, BioTechniques Books, Natlck,Mass.; Schmidt and Mueller, 1999, Nucleic Acids Res. 27, 331).

For the PCR reaction, two sets of primers were used for each testedRNA-3′-specific/5′-specific primers and 3′-specific/tag-specific primers(FIG. 15).

To determine the possibility of using the method of chemical ligation ofRNA with tag known sequence specifically to the cap-structure of viralRNAs, the genomic RNA of tobacco mosaic virus (TMV) U1 strain which isknown to be capped (Dunigan and Zaitlin, 1990, J. Biol. Chem. 265,7779-7786.) was used as control. The respective PCR bands were detectedwhen specific primers, U1-Spn and corresponding to RNA-tag primer 779were used in the PCR reaction (Table 2, FIG. 16). TABLE 2 Templates andprimers used for PCR. Corresponding PCR Forward band and detectionTemplate primer Reverse primer of cap-structrure Genomic TMV (U1) RNAU1-Spn + Genomic TMV (U1) RNA 779 U1-Spn + (cap) Non-capped RNAtranscript of TMV U1-Spn + Non-capped RNA transcript of TMV 779 U1-Spn −(non-capped) Complete cDNA clone of TMV (U1) U1-Spn + Genomic crTMV RNAK5 2PM + Genomic crTMV RNA 779 2PM + (? - cap?) Non-capped RNAtranscript of crTMV K5 2PM + Non-capped RNA transcript of crTMV 779 2PM− (non-capped) Complete cDNA clone of crTMV K5 2PM + Subgenomic TMV (U1)RNA for MP 2211 UM50-54 + Subgenomic TMV (U1) RNA for MP 779 UM50-54 −(non-capped) Complete cDNA clone of TMV (U1) 2211 UM50-54 + SubgenomiccrTMV RNA for MP 1038 CPF25 + Subgenomic crTMV RNA for MP 779 CPF25 −(non-capped) Complete cDNA clone of crTMV 1038 CPF25 0

As a control, the non-capped RNA-transcript of the complete cDNA cloneof TMV (U1) was used, and the cap structure was not found as expected(Table 2, FIG. 16).

Then the presence of a cap structure at the 5′-terminus of the genomicRNA of crTMV was tested. For these experiments, the specific PCR primersK5, 2PM and primer 779 which corresponds to the RNA-tag were taken(Table 1, FIG. 16). Interestingly, the mobility of the PCR band observedwith the primers 779 and 2PM, was higher than expected (FIG. 16). Thiscould reflect the presence of a strong secondary structure at the5′-terminus of the genomic RNA of crTMV (Dorokhov et al., 1994, FEBSLetters 350, 5-8). This secondary structure is absent at the 5′-terminalpart of related TMVs (Goelet et al., 1982, Proc. Natl. Acad. Sci. USA79, 5818-5822). In control experiments with non-capped transcript of thecomplete cDNA clone of crTMV, no respective PCR band was observed, asexpected.

For subgenomic RNA coding for the TMV (U1) MP gene, the absence of acap-structure at the 5′-terminus was proposed. We tested the respectivesgRNA with the specific primers 2211, UM50-54 and primer 779corresponding to the RNA-tag. No cap structure was found (Table 2, FIG.16).

The same results were obtained with the respective subgenomic RNA ofcrTMV (Table 2, FIG. 16) indicating that cap-structure is absent at the5′-terminus of this subgenomic RNA of tobamoviruses.

Insertion of IRES_(MP,75) ^(CR) into a TMV UI Based Vector that isDeficient of MP Gene Expression, KK6 Provides Efficient Cap-IndependentMP Gene Expression

The KK6 vector (Lehto et al., 1990, Virology 174, 145-157) contains twoCP subgenomic promoters (sgPr). The first CP sgPr-1 is in its properplace, upstream of the CP gene, whereas the second, CP sgPr-2 is placedupstream of the MP gene. It was shown that the MP gene was expressed viaCP sgPr-2 instead of native MP sgPr. As a result of this insertion, KK6lost the capability of efficient cell-to-cell movement. Analysis showedthat I2 sgRNA does not contain an IRES_(MP,75) ^(CR) element in its5′-nontranslated leader. It has been proposed that IRES_(MP,75)^(CR)-lacking KK6 I₂ sgRNA cannot express the MP gene efficiently. Inorder to examine this suggestion, IRES_(MP,75) ^(CR) was inserted intoKK6 between the CP sgPr-2 and the MP gene and we were able to obtainKK6-IRES_(MP75) that was stable in progeny (FIG. 17). It was shown thatKK6-IRES_(MP75) provides synthesis of I₂ sgRNA containing crTMVIRES_(MP75) (FIG. 18).

It can be seen that the start of KK6-IRES_(MP75) I₂ sgRNA is not changedin comparison to KK6, which means that IRES_(MP75) does not serve as MPsgPr.

This insertion drastically improved cell-to-cell movement. KK6 infectedSamsun plants systemically but the first symptoms developed slowly(15-17 days) compared to those induced by wild-type TMV (TMV 304) (about7 days). Symptoms in the upper leaves of KK6-infected plants weredistinct: yellow spots in contrast to mosaic symptoms were produced bywild-type TMV.

KK6 virus progeny produced numerous lesions in N. glutinosa thatdeveloped slower than lesions induced by wild-type TMV UI. The averagesize of local lesions induced by KK6 was approximately 0.1 mm incomparison to those induced by TMV UI (1.1 mm).

Plants inoculated by KK6-IRES_(MP75) looked like KK6-infected Samsunplants but: (i) the first systemic symptoms were developed more rapidly(about 10 days) and (ii) they were much brighter including yellow spotsand mosaic. In contrast to KK6 the average size of local lesions inducedby K86 in N. glutinosa was increased to 0.6-0.7 mm. Examination of thetime-course of MP accumulation showed that KK6-IRES_(MP75) MP isdetected earlier than KK6 MP in inoculated leaves (FIG. 19). Theseresults allowed the conclusion that insertion of IRES_(MP75) ^(CR)upstream of the KK6 MP gene partially restores the movement propertiesof KK6 defective in cell-to-cell and long-distance transport.

In order to obtain additional evidences of the essential role of IRES incap-independent MP gene expression of TMV cDNA vectors and in the lifecycle of tobamoviruses, series of additional KK6-based vectors wasconstructed (FIG. 17). KK6-IRES_(MP125) contains a natural hairpin-loopstructure which is able to inhibit translation of the MP gene in vitroin the presence of WT crTMV 5′leader of I₂ sgRNA (FIG. 13) andIRES_(MP75). KK6-H-PL contains a natural hairpin-loop structure and a72-nt artificial polylinker sequence. KK6-PL contains the polylinkerregion only. Results of tests for infectivity on Nicotiana tabacum cv.Samsun plants (systemic host) are presented in Table.3.

FIG. 20 shows the results of a Western test of CP accumulation intobacco leaves infected with KK6-based vectors. Replacement ofIRES_(MP75) ^(CR) by a nonfunctional PL-sequence drastically blockedvector multiplication. TABLE 3 Virus accumulation in tobaccosystemically infected by KK6-based vectors. cDNA copies Virusaccumulation TMV 304 (WT) +++ KK6 + KK6-IRES_(MP75) ++ KK6-IRES_(MP125)++ KK6-H-PL +/− KK6-PL +/−

EXAMPLE 5

Creation of Artificial, Non-Natural IRES Elements Without SubgenomicPromoter Activity Provides Cap-Independent Expression of Genes ofInterest in Eukaryotic Cells

The goal of this example is to demonstrate the approaches for creationof artificial, non-natural IRES elements free of subgenomic promoteractivity, which provide cap-independent expression of a gene of interestin eukaryotic cells.

Construction of an Artificial, Non-Natural IRES Element on the Basis of18-nt Segment of IRES_(MP,75) ^(CR)

Analysis of the IRES_(MP,75) ^(CR) nucleotide sequence shows that it hasa multimer structure and contains four nucleotide sequence segmentsbeing a variation of element (−72) GUUUGCUUUUUG(−61) and having highcomplementarity to A. thaliana 18S rRNA (FIG. 21). In order to design anartificial, non-natural IRES, the 18-nt sequence CGUUUGCUUUUUGUAGUA wasselected.

Four oligos were synthesized: MP1(+):5′-CGCGCAAGCTTTGCTTTTTGTAGTACGTTTGCTTTTTGTAGTACTGCAGGCGGG-3′ MP1(−):5′-CCCGCCTGCAGTACTACAAAAAGCAAACGTACTACAAAAAGCAAAGCTTGCGCG-3′ MP2(+):5′-GGCGGCTGCAGTTTGCTTTTTGTAGTACGTTTGCTTTTTGTAGTAGAATTCGG-GC-3′ MP2(−):5′-GCCCGAATTCTACTACAAAAAGCAAACGTACTACAAAAAGCAAACTGCAGCCG-CC-3′

Primers MP1(+) and MP1(−) were annealed to each other yelding dsDNAfragment A: CGCGCAAGCTTTGCTTTTTGTAGTACGTTTGCTTTTTGTAGTACTGCAGGCGGGGCGCGTTCGAAACGAAAAACATCATGCAAACGAAAAACATCATGACGTCCGCCC     HindIII                                PstI

Primers MP2(+) and MP2(−) were annealed to each other yelding dsDNAfragment B: GGCGGCTGCAGTTTGCTTTTTGTAGTACGTTTGCTTTTTGTAGTAGAATTCGGGCCCGCCGACGTCAAACGAAAAACATCATGCAAACGAAAAACATCATCTTAAGCCCG       PstI                                   EcoRI

Both fragments were digested with PstI and ligated to each other. Thenthe ligation product A+B was extracted using agarose electrophoresis anddigested with HindIII and EcoRI followed by ligation into the hGFP-GUSvector described by Skulachev et al. (1999, Virology 263, 139-154) usingHindIII and EcoRI cloning sites (FIG. 22).

Results

The transcripts depicted in FIG. 22 were translated in rabbitreticulocyte lysate (RRL) as described by Skulachev et al. (1999,Virology 263, 139-154) and synthesized products were analyzed by gelelectrophoresis. Results represented in FIG. 22 show that an artificial,non-natural sequence based on a 18-nt segment of IRES_(MP,75) ^(CR)provides 3′-proximal-located GUS gene expression. This means that twofeatures, namely complementarity to 18S rRNA and multimer structure areessential for IRES_(MP,75) ^(CR) function and effectiveness.

A tetramer of 18-nt segment does not reach the level of IRES_(MP,75)^(CR) activity but there is a way to improve the activity of artificial,non-natural IRES elements using the 12-nt segment GCUUGCUUUGAG which iscomplementary to 18S rRNA.

Construction of an Artificial, Non-Natural IRES using 19-nt Segment ofIRES_(CP,148) ^(CR)

Analysis of structural elements essential for IRES_(CP,148) ^(CR)activity (FIGS. 23-26) shows that a polypurine (PP) segment is crucialfor IRES_(CP,148) ^(CR) functioning. As a prominent element of the PPtract, a 9-nt direct repeat in 19-nt sequence: AAAAGAAGGAAAAAGAAGG(called direct repeat (DR)) was used for the construction of anartificial IRES. In order to obtain the tetramer of DR the followingprimers were used: CPI(+):5′-CGCGCAAGCTTAAAAGAAGGAAAAAGAAGGAAAAGAAGGAAAAAGAAGGCT- GCAGGCGGG-3′CP1(−):5′-CCCGCCTGCAGCCTTCTTTTTCCTTCTTTTCCTTCTTTTTCCTTCTTTTAAGCT-TGCGCG- 3′CP2(+): 5′-GGCGGCTGCAGAAAAGAAGGAAAAAGAAGGAAAAGAAGGAAAAAGAAGGAA-TTCGGGC-3′ CP2(−):5′-GCCCGAATTCCTTCTTTTTCCTTCTTTTCCTTCTTTTTCCTTCTTTTCTGCAGC-CGCC-3′

According to the experimental procedure described above, the followingIRES element was used as intercistronic spacer:5′-CGCGCAAGCUUAAAAGAAGGAAAAAGAAGGAAAAGAAGGAAAAAGAAGGCU-GCAGAAAAGAAGGAAAAAGAAGGAAAAGAAGGAAAAAGAAGGAAUUCAUG-3′Results

The transcripts depicted in FIG. 22 were translated in rabbitreticulocyte lysate (RRL) as described by Skulachev et al. (1999,Virology 263, 139-154) and synthesized products were analyzed by gelelectrophoresis. The results represented in FIG. 22 show that anartificial, non-natural sequence based on repeated 19-nt segment ofIRES_(CP,148) ^(CR) provides the efficient expression of a 3′-proximallylocated GUS gene.

EXAMPLE 6

Construction of a TMV cDNA Transcription Vector Expressing a ReplicaseGene in Infected Cells in a Cap-Independent Manner

The main goal of this example was to obtain two new TMV U1-based viruseswith modified 5′UTR providing expression of the replicase gene in acap-independent manner:

1) Omega-leader of TMV was completely substituted by IRES_(MP,75) ^(CR).GUUCGUUUCGUUUUUGUAGUAUAAUUAAAUAUUUGUCAGAUAAGAGAUUGGUUAGAGAUUUGUUCUUUGUUUGACCAUGG.

2) Since it is believed that the first 8 nucleotides of the TMV 5′UTRare essential for virus replication (Watanabe et al., 1996, J. Gen.Virol. 77, 2353-2357), IRES_(MP,75) ^(CR) was inserted into TMV leavingthe first 8 nucleotides intact:GUAUUUUUGUAGUAUAAUUAAAUAUUUGUCAGAUAAGAGAUUGGUUAGAGAUUUGUUCUUUGUUUGACCAUGG.

The following primers were used:

-   -   a) SP6-IRES-1 (in the case of the first variant)

Xbal SP6 Promotor IRES_(MP,75) ^(CR) GGGTCTA GATTTAGGTGACACTATAGTTCGTTTCGTTTTTGTAGTA

-   -   b) SP6-IRES-2 (in the case of the second variant)

Xbal SP6 Promotor IRES_(MP,75) ^(CR) GGGTCTA GATTTAGGTGACACTATAGTATTTTTGTAGTATAATTAAATATTTGTC.

c) IRES-NcoI (reverse primer to obtain IRES with a NcoI site at 3′end):GGGCCATGGTCAAACAAAGAACAAATCTCTAAAC.

d) TMV-NcoI (direct primer to obtain TMV polymerase, starting from NcoIsite):     NcoI GGGCCATGGCATACACACAGACAGCTAC.

e) TMV-Xho (reverse primer to obtain 5′-part of replicase from AUG toSphI site) XhoI ATGTCTCGAGCGTCCAGGTTGGGC.Cloning Strategy:

PCR fragment A was obtained using oligos SP6-IRES1 and IRES-NcoI andcrTMV clone as template. PCR fragment B was obtained using oligosTMV-NcoI and TMV-XhoI and TMV-304L clone. Fragments A and B were clonedsimultaneously into the pBluscriptSK+vector using Xbal and XhoI sites(fragments were ligated together through NcoI site). The same procedurewas applied to obtain the second variant of the virus using SP6-IRES2oligo. At the next stage, the whole TMV cDNA was cloned into theobtained vector using SphI and KpnI sites to restore the viral genome(FIG. 27).

EXAMPLE 7

Construction of Tobamoviral Vectors Act2/crTMV and Act2/crTMVIRES_(MP,75) ^(CR)-GUS Based on Actin 2 Transcription Promoters

The main goal of this example is the demonstration of the constructionstrategy of a new crTMV-based vector with which viral genome expressionin plant cells occurs under the control of an efficient Actin 2transcription promoter. It allows the use of the vectorAct2/crTMV/IRES_(MP,75) ^(CR)-GUS for gene expression in plants.

Cloning Act2 into pUC19

The Act2 transcription promoter (about 1 000 bp) was cut out of plasmidpACRS029 by digestion with KpnI and Pst and cloned into pUC19 digestedwith KpnI and PstI.

Creation of a PstI site in Plasmid T7/crTMV (see FIG. 10) Upstream ofcrTMV Genome Start

334-nt cDNA fragment of the 5′-terminal portion of the crTMV genomeobtained by PCR using the direct primerATGCTGCAGGTTTTAGTTTTATTGCAACAACAA (the PstI site is underlined) and thereverse primer ATGCGATCGAAGCCACCGGCCMGGAGTGCA (PvuI site is alsounderlined) was digested with PvuI and PstI and inserted into pUC19Act2together with the part of crTMV genome (PvuI-SpeI fragment).

Cloning of the Rest of the Genome Together with the Last Construct

The Act2 containing construct was inserted into plasmid T7/crTMV afterdigestion with KpnI/SpeI.

Fusion of 5′-Terminus of crTMV to Act2 Transcriptional Start withoutAdditional Sequences

This step was carried out by site-directed mutagenesis usingoligonucleotide primer specific for both Act2 and crTMV to obtain thefinal construct Act2/crTMV (FIG. 28).

To get the vector Act2/crTMV/IRES_(MP,75) ^(CR)-GUS (FIG. 29) theSpeI-NotI cDNA fragment of plasmid Act2/crTMV (FIG. 28) was replaced bythe SpeI-NotI DNA fragment of T7/crTMV/IRES_(MP,75) ^(CR)-GUS construct(FIG. 11) that contains the GUS gene under the control of IRES_(MP,75)^(CR).

EXAMPLE 8

Construction of Circular Single-Stranded Tobamoviral VectorKS/Act2/crTMV/IRES_(MP,75) ^(CR)-GUS (FIG. 30)

The main goal of this example is to demonstrate the possibility of usingcircular single-stranded DNA vectors for foreign gene expression inplants.

In order to construct KS/crTMV/IRES_(MP,75) ^(CR)-GUS (FIG. 30), 9.2 kbKpnI-NotI cDNA fragment of vector Act2/crTMV/IRES_(MP,75) ^(CR)-GUS wasinserted into plasmid pBluescript II KS+ (Stratagene) digested withKpnI-SalI and containing the phage f1 replication origin.Single-stranded DNA of vector KS/Act2/crTMV/IRES_(MP,75) ^(CR)-GUS wasprepared according to Sambrook et al., 1989 (Molecular Cloning: aLaboratory Manual, 2ed edn. Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.).

EXAMPLE 9

Construction of Tobamoviral Vector KS/Act2/crTMV-Int/IRES_(MP,75)^(CR)-GUS Containing Oleosin Intron from Arabidonsis thaliana

The main goal of this example is to create vectorKS/Act2/crTMV/IRES_(MP,75) ^(CR)-GUS containing Arabidopsis thalianaoleosin gene intron that should be removed after transcript processing(FIG. 31).

The cloning strategy comprised the following steps: ps 1. Cloning of A.thaliana Oleosin Gene Intron.

A. thaliana oleosin gene intron was obtained by PCR using A. thalianagenomic DNA and specific primers: A.th./Int (direct)ATGCTGCAGgttttagttCAGTAAGCACACATTTATCATC (PstI site is underlined,lowercase letters depict crTMV 5′terminal sequence) and A.th/Int(reverse) ATGAGGCCTGGTGCTCTCCCGTTGCGTACCTA (StuI is underlined).

2. Insertion of A. thaliana Oleosin Gene Intron into 334-nt 5′-TerminalFragment of crTMV cDNA.

cDNA containing A. thaliana oleosin gene intron was digested withPstI/StuI and ligated with DNA fragment obtained by PCR using primerscorresponding to positions 10-334 of crTMV genome:atgAGGCCTTTATTGCAACAACAACAACAAATTA (StuI site is underlined) andATGCGATCGAAGCCACCGGCCAAGGAGTGCA (PvuI site is underlined).

The next steps were as described in EXAMPLE 7.

EXAMPLE 10

Influence of Rapamycin as an Inhibitor of Cap-Dependent Initiation ofTranslation on GUS Gene Expression in Tobacco Protoplasts transfectedwith IRES_(MP,75) ^(CR) Containing Bicistronic Transcription Vectors,35S/CP/IRES_(MP,75) ^(CR)/GUS (FIG. 32) and 35S/GUS/ IRES_(MP,75)^(CR)/CP (FIG. 33)

The aim of this example is to demonstrate the principal possibility touse inhibitors of cap-dependent translation to increase efficiency ofIRES-mediated cap-independent translation of a gene of interest.

Rapamycin as an inhibitor of cap-dependent initiation of translation wasselected. Recently, a novel repressor of cap-mediated translation,termed 4E-BPI (elF-4E binding protein-1) or PHAS-1 was characterized(Lin et al., 1994, Science 266, 653-656; Pause et al., Nature 371,762-767). 4E-BP1 is a heat- and acid-stable protein and its activity isregulated by phosphorylation (Lin et al., 1994 Science 266, 653-656;Pause et al., Nature 371, 762-767). Interaction of 4EBP1 with elF-4Eresults in specific inhibition of cap-dependent translation, both invitro and in vivo (Pause et_al., Nature 371, 762-767). It has been shownthat rapamycin induces dephosphorylation and consequent activation of4E-BP1 (Beretta et al., 1996, EMBO J. 15, 658-664).

Construction of IRES- and GUS gene-containing vectors 35S/CP/IRES_(MP,75) ^(CR)/GUS (FIG. 32), 35S/GUSI IRES_(MP,75) ^(CR)/CP (FIG.33) and a method of tobacco protoplast transfection with 35S-based cDNAwere described by Skulachev et al. (1999, Virology 263, 139-154).Comparison of GUS gene expression in tobacco protoplats treated byrapamycin and transfected with bicistronic cDNA with GUS gene in 3′- and5′-proximal location shows the possibility to increase IRES-mediatedcap-independent translation of the GUS gene.

EXAMPLE 11

Influence of Potwirus VPg as a inhibitor of Cap-Dependent Initiation ofTranslation on GUS Gene in Tobacco Protoplasts Transfected withIRES_(PM,75) ^(CR) Containing Bicistronic Transcription Vectors35S/CP/IRES_(MP,75) ^(CR)/GUS (FIG. 32) and 35S/CP-VPg/IRES_(MP,75)^(CR)/GUS

This example demonstrates the principal possibility of using a geneproduct to inhibit cap-dependent translation (FIG. 34). Recently,interaction between the viral protein linked to the genome (VPg) ofturnip mosaic potyvirus (TuMV) and the eukaryotic translation initiationfactor elF(iso)4E of Arabidopsis thaliana has been reported (Wittman etal., 1997, Virology 234, 84-92). Interaction domain of VPg was mapped toa stretch of 35 amino acids and substitution of an aspartic acid residuewithin this region completely abolished the interaction. The capstructure analogue m⁷GTP, but not GTP, inhibited VPg-elF(iso)4E complexformation, suggesting that VPg and cellular mRNAs compete for elF(iso)4Ebinding (Leonard et al., 2000, J. Virology 74, 7730-7737).

The capability of VPg to bind elF(iso)4E could be used for inhibition ofcap-dependent translation. We propose to use the vector35S/CP-VPg/IRES_(MP,75) ^(CR)/GUS (FIG. 34) wherein CP is fused with VPgfrom potyvirus potato virus A. Comparison of GUS gene expression inprotoplasts transfected with 35S/CP-VPg/IRES_(MP,75) ^(CR)/GUS or35S/CP/IRES_(MP,75) ^(CR)/GUS would allow to increase IRES-mediated andcap-independent GUS gene expression.

EXAMPLE 12

In vivo Genetic Selection of an IRES Sequence or a Subqenomic Promoterusing TMV Vector

This example demonstrates the possibility of using in vivo geneticselection or Systematic Evolution of Ligands by Exponential enrichment(SELEX) of a subgenomic promoter or an IRES sequence providingcap-independent expression of a gene of interest in a viral vector. Thisapproach proposes using side-by-side selection from a large number ofrandom sequences as well as sequence evolution (Ellington and Szostak,1990, Nature 346, 818-822; Tuerk and Gold, 1990, Science 249, 505-510;Carpenter and Simon, 1998, Nucleic Acids Res. 26, 2426-2432). Theproject encompasses:

-   -   In vitro synthesis of crTMV-based defective-interfering (DI)        transcript containing the following elements (5′-3′        direction): (i) a T7 transcription promoter, (ii) a 5′-terminal        part of crTMV genome with a sequence responsible for viral        genome complementary (minus chain) synthesis, (iii) a sequence        coding for the N-terminal part of a viral replicase, (iv) a        sequence containing 75-nt randomized bases, (v) a neomycin        phosphotransferase 11 (NPT II) gene, (vi) a crTMV origin of        assembly (Oa), and (vii) a 3′-terminal part of the crTMV genome        with minus chain genome promoter sequence (FIG. 35).    -   Co-transfection of tobacco protoplasts by a transcript together        with crTMV genomic RNA (FIG. 10). Protoplasts will grow and        regenerate in media containing kanamycin. Selection and        isolation of an IRES or a subgenomic promoter element providing        protoplast survival and regeneration in the presence of        kanamycin.

ANNEX B Processes and Vectors for Producing Transgencic Plants FIELD OFTHE INVENTION

The present invention relates to processes and vectors for producingtransgenic plants as well as plant cells obtained thereby.

BACKGROUND OF THE INVENTION

Achievement of a desirable and stably inheritable pattern of transgeneexpression remains one of the major problems in plant biotechnology. Thestandard approach is to introduce a transgene as part of a fullyindependent transcription unit in a vector, where the transgene is undertranscriptional control of a plant-specific heterologous or a homologouspromoter and transcription termination sequences (for example, see U.S.Pat. No. 05,591,605; U.S. Pat. No. 05,977,441; WO 0053762 A2; U.S. Pat.No. 05,352,605, etc). However, after the integration into the genomicDNA, because of random insertion of exogenous DNA into plant genomicDNA, gene expression from such transcriptional vectors becomes affectedby many different host factors. These factors make transgene expressionunstable, unpredictable and often lead to the transgene silencing inprogeny (Matzke & Matzke, 2000, Plant Mol Biol., 43, 401-415; S. B.Gelvin, 1998, Curr. Opin. Biotechnol., 9, 227-232; Vaucheret et al.,1998, Plant J., 16, 651-659). There are well-documented instances oftransgene silencing in plants, which include the processes oftranscriptional (TGS) and posttranscriptional gene silencing (PTGS).Recent findings reveal a close relationship between methylation andchromatin structure in TGS and involvement of RNA-dependentRNA-polymerase and a nuclease in PTGS (Meyer, P., 2000, Plant Mol.Biol., 43 221-234; Ding, S. W., 2000, Curr. Opin. Biotechnol., 11,152-156; lyer et al., Plant Mol. Biol., 2000, 43, 323-346). For example,in TGS, the promoter of the transgene can often undergo methylation atmany integration sites with chromatin structure not favorable for stabletransgene expression. As a result, practicing existing methods requiresmany independent transgenic plants to be produced and analyzed forseveral generations in order to find those with the desired stableexpression pattern. Moreover, even such plants displaying a stabletransgene expression pattern through the generations can becomesubsequently silenced under naturally occurring conditions, such as astress or pathogen attack. Existing approaches aiming at improvedexpression control, such as use of scaffold attachment regions (Allen,G. C., 1996, Plant Cell, 8, 899-913; Clapham, D., 1995, J. Exp. Bot.,46, 655-662; Allen, G. C., 1993, Plant Cell, 5, 603-613) flanking thetranscription unit, could potentially increase the independency andstability of transgene expression by decreasing dependency fromso-called “position effect variation” (Matzke & Matzke, 1998, Curr.Opin.Plant Biol., 1, 142-148; S. B. Gelvin, 1998, Curr. Opin. Biotechnol., 9,227-232; WO 9844 139 A1; WO 006757 A1; EP 1 005 560 A1; AU 00,018,331A1). However, they only provide a partial solution to the existingproblem of designing plants with a required expression pattern of atransgene.

Gene silencing can be triggered as a plant defence mechanism by virusesinfecting the plant (Ratcliff et al., 1997, Science, 276, 1558-1560;Al-Kaff et al., 1998, Science, 279 2113-2115). In non-transgenic plants,such silencing is directed against the pathogen, but in transgenicplants it can also silence the transgene, especially when the transgeneshares homology with a pathogen. This is a problem, especially when manydifferent elements of viral origin are used in designing transcriptionalvectors. An illustrative example is the recent publication by Al-Kaffand colleagues (Al-Kaff et al., 2000, Nature Biotech., 18, 995-999) whodemonstrated that CaMV (cauliflower mosaic virus) infection of atransgenic plant can silence the BAR gene under the control of theCaMV-derived 35S promoter.

During the last years, the set of cis-regulatory elements hassignificantly increased and presently includes tools for sophisticatedspatial and temporal control of transgene expression. These includeseveral transcriptional elements such as various promoters andtranscription terminators as well as translational regulatoryelements/enhancers of gene expression. In general, translation enhancerscan be defined as cis-acting elements which, together with cellulartrans-acting factors, promote the translation of the mRNA. Translationin eukaryotic cells is generally initiated by ribosome scanning from the5′ end of the capped mRNA. However, initiation of translation may alsooccur by a mechanism which is independent of the cap structure. In thiscase, the ribosomes are directed to the translation start codon byinternal ribosome entry site (IRES) elements. These elements, initiallydiscovered in picomaviruses (Jackson & Kaminski, 1995, RNA, 1,985-1000), have also been identified in other viral and cellulareucaryotic mRNAs. IRESs are cis-acting elements that, together withother cellular trans-acting factors, promote assembly of the ribosomalcomplex at the internal start codon of the mRNA. This feature of IRESelements has been exploited in vectors that allow for expression of twoor more proteins from polycistronic transcription units in animal orinsect cells. At present, they are widely used in bicistronic expressionvectors for animal systems, in which the first gene is translated in acap-dependent manner and the second one is under the control of an IRESelement (Mounfford & Smith, 1995, Trends Genet, 4, 179-184;Martines-Salas, 1999, Curr Opin Biotech., 19, 458-464). Usually theexpression of a gene under the control of an IRES varies significantlyand is within a range of 6-100% compared to cap-dependent expression ofthe first one (Mizuguchi et al., 2000, Mol. Ther., 1, 376-382). Thesefindings have important implications for the use of IRESs, for examplefor determining which gene shall be used as the first one in abicistronic vector. The presence of an IRES in an expression vectorconfers selective translation not only under normal conditions, but alsounder conditions when cap-dependent translation is inhibited. Thisusually happens under stress conditions (viral infection, heat shock,growth arrest, etc.), normally because of the absence of necessarytrans-acting factors (Johannes & Sarnow, 1998, RNA, 4, 1500-1513;Sonenberg & Gingras, 1998, Cur. Opin. Cell Biol., 10, 268-275).

Translation-based vectors recently attracted attention of researchersworking with animal cell systems. There is one report connected with theuse of an IRES-Cre recombinase cassette for obtaining tissue-specificexpression of cre recombinase in mice (Michael et al., 1999, Mech. Dev.,85, 35-47). In this work, a novel IRES-Cre cassette was introduced intothe exon sequence of the EphA2 gene, encoding an Eph receptor of proteintyrosine kinase expressed early in development. This work is of specificinterest as it is the first demonstration of the use of translationalvectors for tissue-specific expression of a transgene in animal cellsthat relies on transcriptional control of the host DNA. Anotherimportant application for IRES elements is their use in vectors for theinsertional mutagenesis. In such vectors, the reporter or selectablemarker gene is under the control of an IRES element and can only beexpressed if it inserts within the transcribed region of atranscriptionally active gene (Zambrowich et al., 1998, Nature, 392,608-611; Araki et al., 1999, Cell Mol Biol., 45, 737-750). However,despite the progress made in the application of IRESs in animal systems,IRES elements from these systems are not functional in plant cells.Moreover, since site-directed or homologous recombination in plant cellsis extremely rare and of no practical use, similar approaches with plantcells were not contemplated.

There are significantly less data about plant-specific IRES elements.Recently, however, several IRESs that are also active in plants werediscovered in tobamovirus crTMV (a TMV virus infecting Cruciferaeplants) (Ivanov et al., 1997, Virology, 232, 32-43; Skulachev et al.,1999, Virology, 263, 139-154; WO 98/54342) and there are indications ofIRES translation control in other plant viruses (Hefferon et al., 1997,J. Gen Virol., 78, 3051-3059; Niepel & Gallie, 1999, J. Virol., 73,9080-9088). IRES technology has a great potential for the use intransgenic plants and plant viral vectors providing convenientalternative to existing vectors. Up to date, the only known applicationof plant IRES elements for stable nuclear transformation is connectedwith the use of IRESs to express a gene of interest in bicistronicconstructs (WO 98/54342). The construct in question comprises, in 5′ to3′ direction, a transcription promoter, the first gene linked to thesaid transcription promoter, an IRES element located 3′ to the firstgene and the second gene located 3′ to the IRES element, i.e., it stillcontains a full set of transcription control elements.

Surprisingly, we have found that translational vectors that are devoidof their own transcription control elements and rely entirely oninsertion into a transcriptionally active genomic DNA of a plant host,allow recovery of numerous transformants which express the gene ofinterest. Even more surprisingly, such transformants could be easilydetected even in host plants with a very low proportion oftranscriptionally active DNA in their genome such as wheat. Thisinvention is the basis of the proposed process that allows for design oftransgene expression that is entirely controlled by the host'stranscriptional machinery, thus minimizing the amount of xenogenetic DNAelements known to trigger transgene silencing. It also allows to controltransgene expression in a novel way, by modulating the ratio ofcap-dependent versus cap-independent translation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 36 shows transgene expression from four of many possibletranslational vector variants.

A—the vector contains a translation enhancer and a translation stopcodon;

B—the vector contains an IRES as translation enhancer and atranscription termination region;

C—as in B, except that the IRES is preceded by translation stop codonsfor all three reading frames;

D—as in C, except that the vector is flanked by intron/exon boundaryregions (3′I-5′E and 3′E-5′I) to provide the features of an exon and tofacilitate its incorporation into the spliced mRNA.

FIG. 37 depicts vector pIC1301 containing IRES_(MP,75) ^(CR), BAR andthe 35S terminator.

FIG. 38 depicts vector pIC1521 containing a “hairpin”, IRES_(CP,148)^(CR), BAR and the 35S terminator. The “hairpin” structure serves as analternative to the translation stop codon, preventing the formation ofthe translational fusion products.

FIG. 39 depicts vector pIC1451 containing a promoterless BAR gene andthe 35S terminator.

FIG. 40 depicts vector pIC052 containing loxP, HPT and nos terminator.

FIG. 41 depicts vector pIC06-IRES containing IRES_(MP,75) ^(CR), theAHAS gene, whereby AHAS is the mutated version of the Arabidopsisacetohydroxyacid synthase gene conferring resistance to imidazolineherbicides.

FIG. 42 depicts vectors pIC-DOG and pIC-CRE containing the codingsequence of the yeast 2-deoxyglucose-6-phosphate (2-DOG-6-P) phosphataseand cre recombinase under the control of the rice actin promoter,respectively.

FIG. 43 depicts the transposon-incorporated translational vectorpIC-dSpm and vector pIC1491 containing a transposase. PHBT is achimaeric promoter consisting of p35S enhancers fused to the basal partof the wheat C4PPDK gene.

DETAILED DESCRIPTION OF THE INVENTION

A primary objective of this invention is to provide a novel process orvector to produce transgenic plants for the stable expression oftransgenic material integrated into a plant genome.

This object is achieved by a process for producing transgenic plants orplant cells capable of expressing a transgenic coding sequence ofinterest under transcriptional control of a host nuclear promoter byintroducing into the nuclear genome a vector comprising in itstranscript a sequence for binding a plant cytoplasmic ribosome in a formfunctional for initiation of translation and, downstream thereof, saidtransgenic coding sequence, and subsequently selecting plant cells orplants expressing said transgenic coding sequence. The gene of interestis under control of a translation signal, such as but not limited to, anIRES element and it has no promoter operably linked to it. Such vectorsrely on transgene insertions into transcriptionally active DNA of thehost genome.

Further a novel vector is provided for transforming plant cells,comprising, optionally after processing in the host cell, in itstranscript a sequence for binding a plant cytoplasmic ribosome in a formfunctional for the initiation of translation and, downstream thereof, acoding sequence, said vector being devoid of a promoter functional forthe transcription of said coding sequence.

Preferred embodiments are defined in the subclaims.

Construction of vectors for stable transformation of plants has beendescribed by numerous authors (for review, see Hansen & Wright, 1999,Trends in Plant Science, 4, 226-231; Gelvin, S. B., 1998, Curr. Opin.Biotech., 9, 227-232). The basic principle of all these constructs isidentical—a fully functional transcription unit consisting of, in 5′ to3′direction, a plant-specific promoter, a structural part of a gene ofinterest and a transcriptional terminator, has to be introduced into theplant cell and stably integrated into the genome in order to achieveexpression of a gene of interest.

We have developed a different technology for obtaining stable nucleartransformants of plants. Our invention relies on the surprising findingthat the host plant transcription machinery is able to drive theformation of mRNA from a transgene of interest in a transformed plantcell. The proposed process utilizes vectors having a gene of interestthat is not operationally linked to a promoter in said vector. Rather,they comprise the coding region of a gene of interest under the controlof translation elements only. Said translational element may be asequence for binding, preferably after transcription, a plantcytoplasmic ribosome thus enabling translation of a coding sequencedownstream thereof. Preferably, said translational element is a ribosomeentry site functional in plants and more preferably a plant-specificIRES element, notably an IRES element of plant viral origin, of plantorigin, of non-plant origin or an artificially designed IRES element.

Such a vector DNA, after integration into the transcribed region of aresident plant gene, yields chimaeric mRNA and is subsequentlytranslated into the protein of interest via initiation of translationfrom said sequence for binding a plant cytoplasmic ribosome (FIG. 36).To the best of our knowledge, there is no prior art concerning thisapproach for generating stable nuclear plant transformants. It was verysurprising, that, given the low proportion of transcriptionally activeDNA in most plant genomes, transformation experiments utilizingtranslation vectors described in the present invention, yielded numeroustransformants expressing the gene of interest.

Our invention addresses imminent problems of reliable transgeneexpression. The transgene integrated into host genome using our inventedprocess, relies on the transcription machinery including all or most ofthe transcriptional regulatory elements of the host's resident gene,thus minimizing transgene silencing usually triggered by xenogenetic DNAelements.

The vectors for transgene delivery can be built in many different ways.The simplest versions consist only of the coding region of a gene ofinterest or a portion thereof with a translation signal (basictranslational vector). In a preferred vector, a translational stopsignal is provided upstream of said sequences for binding a plantcytoplasmic ribosome. The stop signal may for example be at least onestop codon and/or an RNA hairpin secondary structure or the like. Thisstop signal causes abortion of upstream translation. More advancedversions may include a plant-specific IRES element followed by thecoding region (of a gene) of interest. Advanced versions of thetranslational vector may include sequences for site-specificrecombination (for review, see Corman & Bullock, 2000, Curr OpinBiotechnol, 11, 455-460) allowing either the replacement of an existingtransgene or integration of any additional gene of interest into thetranscribed region of the host DNA. Site-specificrecombinases/integrases from bacteriophages and yeasts are widely usedfor manipulating DNA in vitro and in plants. Examples forrecombinases-recombination sites for the use in this invention includethe following: cre recombinase-LoxP recombination site, FLPrecombinase-FRT recombination sites, R recombinase-RS recombinationsites, phiC31 integrase—attP/attB recombination sites etc.

The introduction of splicing sites into the translation vector may beused to increase the probability of transgene incorporation into theprocessed transcript.

The vector may further comprise a sequence coding for a targeting signalpeptide between said sequence for binding a plant cytoplasmic ribosomeand said coding sequence. Preferable examples of such signal peptidesinclude a plastid transit peptide, a mitochondrial transit peptide, anuclear targeting signal peptide, a vacuole targeting peptide, and asecretion signal peptide.

Various methods can be used to deliver translational vectors into plantcells, including direct introduction of said vector into a plant cell bymeans of microprojectile bombardment, electroporation or PEG-mediatedtreatment of protoplasts. Agrobacterium-mediated plant transformationalso presents an efficient way of the translational vector delivery. TheT-DNA insertional mutagenesis in Arabidopsis and Nicotiana with thepromoterless reporter APH(3′)II gene closely linked to the right T-DNAborder showed that at least 30% of all inserts induced transcriptionaland translational gene fusions (Koncz et al., 1989, Proc. Natl. Acad.Sci., 86, 8467-8471).

A translational vector can also be cloned into transposable elements,facilitating the search for suitable transcribed regions and providingeither a constitutive or tissue/organ-specific pattern of transgeneexpression. Transposable elements are extensively used in plants withthe purpose of inactivation-based gene tagging (Pereira & Aarts, 1998,Methods Mol Biol., 82, 329-338; Long & Coupland, 82, 315-328; Martin GB., 1998, Curr Opin Biotechnol., 9, 220-226). Different versions of thetransposon-tagging systems were developed. In the simplest version,transposons are used for insertional mutagenesis without anymodifications except, possibly, for deletions or frame-shift mutationsin order to generate non-autonomous transposable elements. In moresophisticated versions, additional genes are inserted into thetransposable elements, e.g. reinsertion markers, reporter genes,plasmid-rescue vectors (Carroll et al., 1995, Genetics, 13, 407-420;Tissier et al., 1999, Plant Cell, 11, 1841-1852). There are so-calledenhancer-trap and gene-trap systems (Sundaresan et al., 1995, GenesDev., 9, 1797-810; Fedorov & Smith, 1993, Plant J., 3, 273-89).Transposable elements in such systems are equipped either with apromoterless reporter gene or a reporter gene under the control of aminimal promoter. In the first case, the reporter gene can be expressedfollowing insertion into the transcribed region of host DNA just afterthe host promoter or insertion into the coding region of the host geneand creation of “in frame” fusion with the host gene transcript.

The chance for successful “in frame” fusion can be significantlyincreased by placing in front of the reporter gene a set of splicingdonor and acceptor sites for all three reading frames (Nussaume et al.,1995, Mol Gen Genet., 249, 91-101). In the second case, transcription ofa reporter gene will be activated from the minimal promoter followinginsertion near the active host promoter (Klimyuk et al., 1995, Mol GenGenet., 249, 357-65). The success of such approaches for transposontagging favors the use of a similar approach for the translationalvectors with IRES elements in front of the gene of interest.

All approaches described above aim at designing a system that places atransgene under expression control of the resident gene in which theinsertion occurred. This might be advantageous for specific tasks andcases. In many other cases, a modified pattern of transgene expressionmight be preferable. For such purposes, the translational vector can beequipped with transcriptionally active elements, such as enhancers whichcan modulate the expression pattern of a transgene. It is known thatenhancer sequences can affect the strength of promoters located as faras several thousand base pairs away (Müller, J., 2000, Current Biology,10, R241-R244). The feasibility of such an approach was demonstrated inexperiments with activation tagging in Arabidopsis (Weigel et al., 2000,Plant Physiol., 122, 1003-1013), where T-DNA-located 35S enhancerelements changed the expression pattern of resident genes, and inenhancer-trap transposon tagging described above. In the latter example,resident gene enhancers determined the expression pattern of thereporter transgene. This approach might be useful, for example, at theinitial stages of plant transformation, or when modulation of thetransgene expression pattern is required after the transformation. Theenhancer sequences can be easily manipulated by means ofsequence-specific recombination systems (inserted, replaced or removed)depending on the needs of the application.

Our approach was to preferably make a set of constructs based ondifferent IRES elements functional in plant cells. The constructscontain IRES elements followed by a plant selectable marker gene and atranscription/translation termination signal. These constructs can beused directly for plant cells transformation after being linearized fromthe 5′ end in front of the IRES sequences or can be cloned into theT-DNA for Agrobacterium-mediated DNA transfer. Another set ofconstructs, serving as controls, contained either a promoterlessselectable gene (a negative control) or a selectable gene under thecontrol of a constitutive promoter functional in monocot and/or dicotcells (a positive control). DNA was transformed into plant cells usingdifferent suitable technologies, such as Ti-plasmid vector carried byAgrobacterium (U.S. Pat. No. 5,591,616; U.S. Pat. No. 4,940,838; U.S.Pat. No. 5,464,763), particle or microprojectile bombardment (U.S. Pat.No. 05,100,792; EP 00444882 B1; EP 00434616 B1). In principle, otherplant transformation methods could be used, such as but not limited to,microinjection (WO 09209696; WO 09400583 A1; EP 175966 B1),electroporation (EP 00564595 B1; EP 00290395 B1; WO 08706614 A1).

The transformation method depends on the plant species to betransformed. Our exemplification includes data on the transformationefficiency for representatives of monocot (e.g. Triticum monococcum) anddicot (e.g. Brassica napus, Orichophragmus violaceous) plant species,thus demonstrating the feasibility of our approach for plant species ofdifferent phylogenetic origin and with different densities oftranscribed regions within a species genome. The transgenic codingsequence in the vector may represent only part of a gene of interest,which gene is reconstructed to a functional length as a result ofsite-directed or homologous recombination. The translation of thesequence of interest is preferably cap-independent. The host may bemodified for inhibiting (or enhancing) cap-dependent translation or forenhancing (or inhibiting) cap-independent translation. This may beaccomplished by treatment with exogenous agents or by including asequence in the vector or said plant, which expression has the desiredeffect.

EXAMPLES Example 1

Construction of IRES Containing Vectors

Series of IRES-mediated expression vectors were constructed usingstandard molecular biology techniques (Maniatis et al., 1982, Molecularcloning: a Laboratory Manual. Cold Spring Harbor Laboratory, N.Y.).Vector pIC1301 (FIG. 37) was made by digesting plasmid pIC501(p35S-GFP-IRES_(MP,75) ^(CR)-BAR-35S terminator in pUC120) with HindIIIand religating large gel-purified fragment. The IRES_(MP,75) ^(CR)sequence represents the 3′ terminal 75 bases of the 5′-nontranslatedleader sequence of the subgenomic RNA of the movement protein (MP) of acrucifer (CR)-infecting tombamovirus.

Vector pIC1521 (FIG. 38) was made following three steps of cloning. Inthe first step pIC1311 was constructed by ligating the largeHindIII-PstI fragment of pIC031 with the small HindIII-NcoI fragment ofpIC032 and the small BspHI-PstI fragment of pIC018. The resultingconstruct pIC1311 (not shown) containing the BAR gene under the controlof the 35S promoter was used as the comparative control in thetransformation experiments. Plasmid pIC1311 was digested withHindIII-NruI and blunt-ended by treatment with Klenow fragment of DNApolymerase I. The large restriction fragment was gel-purified andreligated producing pIC1451 (promoterless BAR-35S terminator; see FIG.39). Ligation of the large SacI-PstI fragment of pIC1451 with the smallSacI-NcoI fragment of pIC033 and the small BspHI-PstI fragment of pIC018produced pIC1521 (FIG. 38). This construct contains a “hairpin” in frontof the IRES_(cp,148) ^(CR) (CP stands for coat protein) element. The“hairpin” structure is formed by the presence of an inverted tandemrepeat formed by KpnI-EcoRI and ClaI-KpnI fragments from the BluescriptII SK+ polylinker sequence.

All vectors were linearized for use in the transformation experiments bydigesting either with SacI (pIC1521; pIC1451) or HindIII (pIC1311;pIC1301) restriction enzyme and gel-purified to separate from undigestedvectors.

Example 2

PEG-Mediated Protoplast Transformation of Brassica napus Isolation ofProtoplasts

The isolation of Brassica protoplasts was based on previously describedprotocols (Glimelius K., 1984, Physiol.Plant, 61, 38-44; Sundberg &Glimelius, 1986, Plant Science, 43, 155-162 and Sundberg et al., 1987,Theor. Appl. Genet., 75, 96-104).

Sterilized seeds (see Appendix) were germinated in 90 mm Petri dishescontaining ½ MS medium with 0.3% Gelrite. The seeds were placed in rowsslightly separated from each other. The Petri dishes were sealed, tiltedat an angle of 45° and kept in the dark for 6 days at 28° C. Thehypocotyls were cut into 1-3 mm long peaces with a sharp razor blade.The blades were often replaced to avoid the maceration of the material.The peaces of hypocotyls were placed into the TVL solution (seeAppendix) to plasmolise the cells. The material was treated for 1-3hours at room temperature. This pre-treatment significantly improves theyield of intact protoplasts. The preplasmolysis solution was replacedwith 8-10 ml of enzyme solution (see Appendix). The enzyme solutionshould cover all the material but should not to be used in excess. Thematerial was incubated at 20-25° C. in dark for at least 15 hours. ThePetri dishes were kept on a rotary shaker with very gentle agitation.

The mixture of protoplasts and cellular debris was filtered through 70mm mesh size filter. The Petri dishes were rinsed with 5-10 ml of W5solution (Menczel et al., 1981, Theor. Appl. Genet., 59, 191-195) (alsosee Appendix) that was also filtered and combined with the rest of thesuspension. The protoplast suspension was transferred to 40 ml sterileFalcon tubes and the protoplasts were pelleted by centrifugation at 120g for 7 min. The supernatant was removed and the pellet of protoplastswas re-suspended in 0.5 M sucrose. The suspension was placed into 10 mlsterile centrifuge tubes (8 ml per tube) and loaded with 2 ml of W5solution. After 10 min of centrifugation at 190 g the intact protoplastswere collected from the interphase with a Pasteur pipette. They weretransferred to new centrifuge tubes, resuspended in 0.5 M mannitol with10 mM CaCl₂ and pelleted at 120 g for 5 min.

PEG Treatment

The protoplasts were resuspended in the transformation buffer (seeAppendix). The protoplast concentration was determined using thecounting chamber and than adjusted to 1-1.5×10⁶ protoplasts/ml. The 100μl drop of this suspension was placed at the lower edge of the tilted6-cm Petri dish and left for a few minutes allowing the protoplasts tosettle. The protoplasts were than gently mixed with 50-100 μl of DNAsolution (Qiagen purified, dissolved in TE at the concentration 1mg/ml). Than 200 μl of PEG solution (see Appendix) was added dropwise tothe protoplasts/DNA mixture. After 15-30 min the transformation buffer(or W5 solution) was added in small aliquots (dropwise) until the dishwas almost filled (˜6 ml). The suspension was left to settle for 1-5hours. Then the protoplast were transferred to the centrifuge tubes,re-suspended in W5 solution and pelleted at 120 g for 5-7 min.

Protoplast Culture and Selection for Transformants

The protoplasts were transferred to the culture media 8 pM (Kao &Michayluk, 1975, Planta, 126, 105-110; also see the Appendix) andincubated at 25° C., low light density, in 2.5 cm or 5 cm Petri disheswith 0.5 ml or 1.5 ml of media, respectively. Protoplast density was2.5×10⁴ protoplasts/ml. The three volumes of fresh 8 pM media withoutany hormones were added right after the first protoplasts division. Thecells were incubated at high light intensity, 16 hours per day.

After 10-14 days the cells were transferred to K3 media (Nagy & Maliga,1976, Z. Pflanzenphysiol., 78, 453-455) with 0.1 M sucrose, 0.13%agarose, 5-15 mg/L of PPT and the hormone concentration four times lessthan in 8 pM medium. To facilitate the transfer to fresh media, thecells were placed on the top of sterile filter paper by carefullyspreading them in a thin layer. The cells were kept at high lightintensity, 16 hours per day. The cell colonies were transferred to Petridishes with differentiation media K3 after their size had reached about0.5 cm in diameter.

Example 3

Transformation of Triticum monococcum by Microproiectile Bombardment

Plant Cell Culture

Suspension cell line of T. monococcum L. was grown in MS2 (MS salts(Murashige & Skoog, 1962 Physiol. Plant, 15, 473-497), 0.5 mg/L ThiamineHCl, 100 mg/L inosit, 30 g/L sucrose, 200 mg/L Bacto-Tryptone, 2 mg/L2,4-D) medium in 250 ml flasks on a gyrotary shaker at 160 rpm at 25° C.and was subcultured weekly. Four days after a subculture the cells werespread onto sterile 50 mm filter paper disks on a gelrite-solidified (4g/L) MS2 with 0.5 M sucrose.

Microprojectile Bombardment

Microprojectile bombardment was performed utilizing the BiolisticPDS-1000/He Particle Delivery System (Bio-Rad). The cells were bombardedat 900-1100 psi, with 15 mm distance from a macrocarrier launch point tothe stopping screen and 60 mm distance from the stopping screen to atarget tissue. The distance between the rupture disk and a launch pointof the macrocarrier was 12 mm. The cells were bombarded after 4 hours ofosmotic pretreatment.

A DNA-gold coating according to the original Bio-Rad's protocol (Sanfordet al., 1993, In: Methods in Enzymology, ed. R. Wu, 217, 483-509) wasdone as follows: 25 μl of gold powder (0.6, 1.0 mm) in 50% glycerol (60mg/ml) was mixed with 5 μl of plasmid DNA at 0.2 μg/μl, 25 μl CaCl₂ (2.5M) and 10 μl of 0.1 M spermidine. The mixture was vortexed for 2 minfollowed by incubation for 30 min at room temperature, centrifugation(2000 rpm, 1 min), washing by 70% and 99.5% ethanol. Finally, the pelletwas resuspended in 30 μl of 99.5% ethanol (6 μl/shot). A new DNA-goldcoating procedure (PEG/Mg) was performed as follows: 25 μl of goldsuspension (60 mg/ml in 50% glycerol) was mixed with 5 μl of plasmid DNAin an Eppendorf tube and supplemented subsequently by 30 μl of 40% PEGin 1.0 M MgCl₂. The mixture was vortexed for 2 min and than incubatedfor 30 min at room temperature without mixing. After centrifugation(2000 rpm, 1 min) the pellet was washed twice with 1 ml of 70% ethanol,once by 1 ml of 99.5% ethanol and dispersed finally in 30 μl of 99.5%ethanol. Aliquots (6 μl) of DNA-gold suspension in ethanol were loadedonto macrocarrier disks and allowed to dry up for 5-10 min.

Plasmid DNA Preparation

Plasmids were transformed into E.coli strain DH10B, maxi preps weregrown in LB medium and DNA was purified using the Qiagen kit.

Selection

For stable transformation experiments, the filters with the treatedcells were transferred onto the solid MS2 medium with the appropriatefilter-sterilized selective agent (150 mg/L hygromycin B (Duchefa); 10mg/L bialaphos (Duchefa). The plates were incubated in the dark at 26°C.

Example 4

Transformation of Orychophracimus violaceus by MicroproiectileBombardment

Preparation of the Suspension Culture

Plants of O. violaceus are grown in vitro on MS medium, 0.3% Gelrite(alternatively, ½ MS, 2% sucrose and 0.8% agar) at 24° C. and 16/8 hoursday/night photoperiod for 3-4 weeks. Four-six leaves (depending of size)were cut into small peaces and transferred to the Magenta box with 30 mlof Callus Inducing Medium (CIM) (see the Appendix). The material waskept for 4-5 weeks at dim light (or in dark) at 24° C. and vigorousagitation. During this period the fresh CIM media was added to keep theplant tissue in the Magenta box covered with liquid. The cells stickingto the wall of the Magenta box were released into the media by vigorousinverting and shaking of the box.

Preparation of Plant Material for Microproiectile Bombardment

The aliquote of cell suspension was carefully placed onto the sterilefilter paper supported by solid CIM media in Petri dish. The Petri dishwith plant material was kept in the dark for 5-7 days. Four hours beforethe procedure, the filter paper with cells was moved to fresh CIM with10% sucrose. Microprojectile bombardment was performed as described inExample 3. Fourteen hours after the bombardment the material wastransferred to CIM with 3% sucrose and kept in the dark.

Selection for Transformants

Two-four days after the bombardment, the filter paper with cells wastransferred to the plate with CIM supplemented with the appropriateselection agent (10-15 μg/ml PPT). Every seven days the material wastransferred to fresh selection media. The plates were kept in the darkand after approximately 6 weeks the plant material was transferred tothe Petri plates with Morphogenesis Inducing Medium (MIM) (see theAppendix) supplemented with the appropriate selection agent (10-15μug/ml PPT). The plates were incubated at high light intensity, 16 hoursday length.

Example 5

Transformation of Triticum monococcum with Promoterless loxP-HPT Gene

The construct pIC052 (FIG. 41) was linearized by digestion with HindIIIrestriction enzyme, gel-purified to separate undigested material andused for the microprojectile bombardment as described above (see EXAMPLE3). The linearized vector contains pUC19 polylinker (57 bp) followed bya loxP site from the 5′ end of the HPT gene. In general, approximately100 bp is located at the 5′ end of translation start codon of HPT gene.Thirty four plates were transformed and after 1.5 months of selection onhygromycin-containing media (EXAMPLE 3), three hygromycin resistantcolonies were recovered. The sequence of the integration sites recoveredby IPCR, confirmed the independency of all three transformants.

Example 6

Transformation of Orychophragmus Leaves with Promoterless IRES_(MP,75)^(CR)-AHAS

Plant acetohydroxyacid synthase (AHAS) is a nuclear encoded, chloroplasttargeted protein which catalyses the first step in the biosynthesis ofthe branched chain amino acids. It is under allosteric control by theseamino acids and can be inhibited by several classes of herbicides. Theconstruct pIC06-IRES was made by replacing the promoter of theArabidopsis AHAS(Ser653-Asn) gene (1.3 Kb PstI-NcoI fragment) in pIC06with the IRES_(MP,75) ^(CR) sequence. The final construct (FIG. 41)contained the mutated version of the Arabidopsis acetohydroxyacidsynthase (AHAS) gene with a single amino acid substitution (Ser653Asn)conferring resistance to the imidazoline herbicide family (Sathasivan,Haughn & Murai, 1991, Plant Physiol., 97, 1044-1050). The plasmid waslinearized by treatment with SalI restriction enzyme and used formicroprojectile bombardment of freshly induced O. violaceous suspensionculture. Leaves of sterile O. violaceous plants were cut onto the smallpeaces and placed in the liquid High Auxin Medium (HAM) (see theAppendix) in Magenta boxes on a rotary shaker to induce suspensionculture. After 7-14 days the suspension culture was transferred to thePetri dishes with Greening Medium (GM) covered by sterile filter paper(see the Appendix). After 3 days the filter paper with the cells wastransferred on GM supplemented with 0.4 M sucrose. After four hours thecells were used for microprojectile bombardment with linearized DNA ofpIC06-IRES, as described in EXAMPLE 3. After 14 hours the filter paperwith cells was transferred to GM, 3% sucrose. Two days later the cellswere transferred to GM with 0.7 μM imazethapyr (AC263, 499 or Pursuit,American Cyanamid). The cells were subcultured every 7-10 days. Putativeevents were identified after approximately four-six weeks and thetransformants were selected under high light intensity, 16 hours perday, on the regeneration medium (RM) with 1-2 μM imazethapyr.

Example 7

Expression of 2-DOG-6-P Gene using Translational Vector

The aim of this example is to demonstrate the possibility ofmanipulation with transgenic plant cells already containingtranslational vector sequences with the sequence-specific recombinationsites.

The hygromycin-resistant T. monococcum cells transformed with vectorpIC052 (EXAMPLE 5) were used for microprojectile co-bombardment with twoplasmids, pIC-DOG and pIC-CRE (FIG. 42). Plasmid pIC-DOG containspromoterless 2-deoxyglucose-6-phosphate (2-DOG-6-P) phosphatase cDNA(patent WO 98/45456) flanked by two loxP sites. Cre-mediated integrationof the 2-DOG-6-P gene into the loxP site of pIC052-containingtransformants leads to the expression of 2-DOG-6-P from a residentpromoter. Such expression confers resistance to 2-deoxyglucose (2-DOG).The resistant colonies were selected as described in EXAMPLE 3, butusing 0.075-0.1% of 2-DOG as the selective agent.

Example 8

Transposon-Incorporated Translational Vector

The aim of this example is to show an alternative way to the directtransformation of directing translational vector to a desiredtranscriptional site in a host genome.

Co-transformation of O. violaceous cells with the constructs shown inFIG. 43 and selection for transformants was performed as described inEXAMPLE 4. The non-autonomous transposable dSpm element contains apromoterless BAR gene preceeded from its 5′ end IRES_(MP,75) ^(CR). Thetransposition induced by Spm transposase facilitates the search fortranscriptionally active regions with a desired expression pattern (inthis case—constitutive) in said host genome, thus increasing the numberof recovered primary transformants. Indeed, the number of transformantswas 3-4 times higher than with the IRES_(MP75) ^(CR)-BAR gene alone(pIC1301, FIG. 37).

Appendix

Seed sterilization

Soak the seeds in 1% PPM solution for at least 2 hours (overnight ispreferable). Wash the seeds in 70% EtOH for 1 minute than sterilize in10% chlorine solution with 0.01% SDS or Tween 20) in 250 ml flask placedon the rotary shaker. Wash the seeds in 0.5 L of sterile water. TVLEnzyme solution  0.3 M sorbitol   1% cellulase R10 0.05 M CaCl₂ × 2H₂O0.2% macerase R10 pH 5.6-5.8 0.1% dricelase dissolved in 8 pM macrosaltwith 0.5 M pH 5.6-5.8 W5 PEG solution 18.4 g/L CaCl₂ × 2H₂O 40% (w/v) ofPEG-2000 in H₂O  9.0 g/L NaCl  1.0 g/L glucose  0.8 g/L KCl pH 5.6-5.8CIM MIM Macro MS Macro MS Micro MS Micro MS Vitamin B5 Vitamin B5 MES 500 mg/L MES  500 mg/L PVP  500 mg/L PVP  500 mg/L Sucrose   30 g/LSucrose   30 g/L 2.4-D   5 mg/L ABA   1 mg/L Kin 0.25 mg/L BA  0.5 mg/LGelrite   3 g/L IAA  0.1 mg/L pH 5.6-5.8 Gelrite   3 g/L pH 5.6-5.8Greening High Auxine Medium (GM) Medium (HAM) Macro MS Macro MS Micro MSMicro MS Vit B5 Vit B5 MES  500 mg/L MES  500 mg/L PVP  500 mg/L PVP 500 mg/L Sucrose   30 g/L Sucrose   30 g/L BA   2 mg/L NAA   5 mg/L Kin 0.5 mg/L Kin 0.25 mg/L NAA  0.1 mg/L BA 0.25 mg/L pH 5.6-5.8 pH 5.6-5.8Regeneration Medium Macro MS Micro MS Vit B5 MES  500 mg/L PVP  500 mg/LSucrose   30 g/L ABA   1 mg/L BA  0.5 mg/L IAA  0.1 mg/L pH 5.6-5.8Hormone solutions were filter sterilized and added to the autoclavedmedia.

1. A process of controlling a biochemical process (II) or biochemicalcascade (III) of interest in a plant, said process comprising: (a)introducing into the nuclear genome of the plant one or more firstheterologous DNA sequences; and (b) infecting the plant with at leastone viral transfection vector containing in its genome one or moresecond heterologous DNA or RNA sequences, thus triggering a process ofinteraction (I) in the plant between (i) one or more first heterologousDNA sequences of the nuclear genome and/or expression products of thefirst heterologous DNA sequences, and (ii) one or more secondheterologous DNA or RNA sequences of the transfection vector and/orexpression products of the second heterologous DNA or RNA sequences, and(iii) optionally one or more externally added low molecular weightcomponents, thus switching on the biochemical process (II) orbiochemical cascade (II) of interest that was not operable prior to saidinteraction.
 2. The process according to claim 1, wherein the process ofinteraction requires an expression product of a first heterologous DNAsequence stably integrated in the nuclear genome of the plant.
 3. Theprocess according to claim 1, wherein said interaction requires anexpression product of a second heterologous DNA or RNA sequence of saidtransfer vector.
 4. The process according to claim 1, wherein theinfection of the plant in step (b) is achieved by an assembled virusparticle or infectious viral nucleic acids, or by activating atransfection process by release of viral nucleic acids that werepreviously incorporated into the plant genome.
 5. The process of claim4, wherein said assembled virus particle or said infectious viralnucleic acid is or comprises RNA.
 6. The process according to claim 1,wherein the infection of the plant in step (b) comprisesAgrobacterium-mediated transfer of nucleic acid sequences into cells ofsaid plant.
 7. The process according to claim 1, wherein a furthervector is introduced in step (b) and wherein a sequence and/or anexpression product of said further vector is involved in said process ofinteraction.
 8. The process according to claim 1, wherein the infectionof the plant in step (b) is achieved by introducing one or more vectorsinto cells of said plant, whereby said vector(s) are adapted to undergoprocessing to generate said viral transfection vector in cells of saidplant.
 9. The process according to claim 1, wherein said process ofinteraction is a viral transfection vector-generated process.
 10. Theprocess according to claim 1, wherein the process of interactioninvolves DNA transposition.
 11. The process according to claim 1,wherein the process of interaction involves DNA recombination.
 12. Theprocess according to claim 11, wherein the biochemical process orcascade of interest comprises expression of a first or second DNA or RNAsequence comprising a promoterless gene in anti-sense orientation whichis placed into sense orientation towards a constitutive promoter in saidprocess of interaction.
 13. The process according to claim 1, whereinthe process of interaction involves recognition of a heterologouspromoter by a heterologous RNA polymerase.
 14. The process according toclaim 13, wherein said first and said second DNA or RNA sequencecomprises a heterologous sequence to be expressed under the control of aheterologous promoter not recognized by a plant RNA polymerase, andtranscription of said sequence to be expressed is switched on byinteraction of said promoter with an RNA polymerase functional therewithand being encoded by said second or said first DNA sequence,respectively.
 15. The process according to claim 14, wherein said RNApolymerase is a bacteriophage RNA polymerase and said heterologouspromoter is a bacteriophage promoter.
 16. The process according to claim1, wherein the process of interaction involves a DNA reaction such asDNA replication, ligation, hybridisation, transcription, or DNArestriction.
 17. The process according to claim 1, wherein the processof interaction involves an RNA reaction such as replication, processing,splicing, reverse transcription, hybridization or translation, oractiviation, inhibition or modification thereof.
 18. The processaccording to claim 1, wherein the process of interaction involves aprotein reaction such as protein folding, assembly, activation,posttranslational modification, targeting, binding, enzymatic activityor signal transduction, or activation, inhibition or modificationthereof.
 19. The process according to claim 1, wherein (i) thebiochemical process or cascade of interest comprises expression of afirst or second DNA sequence separated from its promoter by a DNA insertcapable of preventing transcription of the first or second DNA sequence,and (ii) the process of interaction triggered in step (b) results in theexcision of the DNA insert whereby the first or second DNA sequence isexpressed.
 20. The process according to claim 19, wherein the DNA insertis a non-autonomous transposable element which is excised by atransposase (i) encoded by a second DNA sequence on the viral vector foran insert in the nuclear genome, or (ii) encoded by a first DNA sequencein the nuclear genome for an insert in the viral vector.
 21. The processaccording to claim 19, wherein the DNA insert is flanked byunidirectional sites recognizable by a site-specific DNA recombinase (i)encoded by a second DNA sequence on the viral vector for an insert inthe nuclear genome, or (ii) encoded by a first DNA sequence in thenuclear genome for an insert in the viral vector.
 22. The processaccording to claim 1, wherein transcription of a first or a second DNAsequence is switched on by a heterologous or engineered transcriptionfactor capable of recognizing a heterologous or engineered or chimaericpromoter operably linked to a heterologous gene of interest of saidfirst or second DNA sequence, whereby said promoter is not recognizableby any natural plant transcription factor and said heterologous orengineered transcription factor is encoded by a second or a first DNAsequence, respectively.
 23. The process according to claim 22, whereinthe transcription factor is inducible by an externally applied lowmolecular weight component.
 24. The process according claim 1, whereinsaid first heterologous DNA sequence of step (a) is not of plant viralorigin.
 25. A process of controlling a biochemical process (II) orbiochemical cascade (III) of interest in a plant, said processcomprising: (a) introducing into the nuclear genome of the plant one ormore first heterologous nucleic acid sequences; and (b) infecting theplant with at least one vector containing in its genome one or moresecond heterologous nucleic acid sequences, thus triggering a process ofinteraction (I) in the plant between (i) one or more first heterologousnucleic acid sequences on the nuclear genome and/or expression productsof the first heterologous nucleic acid sequences, and (ii) one or moresecond heterologous nucleic acid sequences of the trarsfection vectorand/or expression products of the second heterologous nucleic acidsequences, and (iii) optionally one or more externally added lowmolecular weight components, whereby a viral transfection vector isgenerated in cells of said plant, thus switching on the biochemicalprocess (II) or biochemical cascade (III) of interest that was notoperable prior to said interaction.
 26. The process of claim 5, whereinthe process of interaction requires an expression product of a firstheterologous DNA sequence stably integrated in the nuclear genome of theplant.
 27. A process of producing a product in a transgenic plantcomprising: (a) introducing into the nuclear genome of the plant one ormore first heterologous DNA sequences: and (b) infecting the plant withat least one viral transfection vector containing in its genome one ormore second heterologous DNA or RNA sequences, thus triggering a processof interaction (I) in the plant between (i) one or more firstheterologous DNA sequences of the nuclear genome and/or expressionproducts of the first heterologous DNA sequences, and (ii) one or moresecond heterologous DNA or RNA sequences of the transfection vectorand/or expression products of the second heterologous DNA or RNAsequences, and (iii) optionally one or more externally added lowmolecular weight components, thus switching on the biochemical process(II) or biochemical cascade (III) of interest that was not operableprior to said interaction, thereby producing the product in thetransgenic plant.
 28. The process of claim 25 further comprising: (a)growing the transgenic plant to a desired stage; (b) infecting the plantwith one or more viral transfection vectors, and optionally contactingthe plant with one or more low molecular weight components, thisswitching on the biochemical process or cascade necessary to produce theproduct, said process or cascade not being operable prior to saidinteraction; and (c) producing the product in the plant. 29.Kit-of-parts for performing the process of claim 1, comprising (i) atransgenic plant or seeds thereof, and (ii) a vector, notably a viraltransfection vector.
 30. The vector for performing step (b) claim
 1. 31.The plant used in the process of claim
 27. 32. The plant used in theprocess of claim 28.